Bispecific Proteins with a Chimeric Scaffold

This application claims priority from GB1902391.0, GB1902370.4, GB1902375.3,

GB1902402.5, GB1902401.7, GB1902398.5, GB1902403.3, GB1902378.7, GB1902393.6, GB1902380.3, GB1902384.5, GB1902394.4, GB1902396.9 and GB1902397.7, all filed 21 Feb 2020, the contents and elements of which are herein incorporated by reference for all purposes.

Field

This invention relates to bispecific proteins and their production and uses.

Background

A priority area in medicine, particularly cancer research, is the expansion of the‘druggable’ proteome, which is currently limited to narrow classes of molecular targets. For example, protein-protein interactions (PPIs) are fundamental to all biological processes and represent a large proportion of potential drug targets, but they are not readily amenable to conventional small molecule inhibition.

Modification of the binding of small protein scaffolds using protein engineering techniques is well-established in the art (see for example Poth et al (2013) Peptide Science 100 5 480;

Lee et al (2014) Nature Comm 5 1 DOI 10.1039/ncom m s4814) .

However, techniques for engineering proteins capable of expanding the druggable proteome have yet to be established.

Summary

The present inventors have found that chimeric proteins capable of binding to two or more target molecules can be generated by displaying peptidyl binding motifs, such as short linear motifs (SLiMs), on a peptidyl scaffold. A peptide ligand comprises a peptidyl binding motif. Peptidyl binding motifs are grafted to peptidyl scaffolds by insertion or substitution of amino acid residues into the scaffold. These chimeric proteins may be useful for example, as single- or multi-function protein therapeutics. In particular, bispecific or hetero-bifunctional chimeric proteins may be useful in promoting the degradation of target molecules via cellular protein degradation pathways.

According to various aspects of the invention, the peptidyl scaffold may be a (i) CKS scaffold, (ii) coiled-coil scaffold, (iii) Affibody scaffold, (iv) trefoil scaffold, (v) PDZ domain scaffold, (vi) ubiquitin or ubiquitin-like domain scaffold, (vii) GB1 scaffold, (viii) VWV scaffold (ix) Fibritin scaffold (x) aPP scaffold, (xi) fibronectin scaffold (xii) Zn finger scaffold, (xiii) SH3 scaffold or (xiv) Cystine knot (CK) scaffold.

A first aspect of the invention is directed to chimeric proteins comprising a CKS (Cycl in dependent kinases regulatory subunit) scaffold.

A first set of embodiments of the first aspect of the invention provide a chimeric protein comprising;

(i) a CKS scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or more of the first loop, second loop, third loop and helical region of the CKS scaffold of the chimeric protein.

In some preferred embodiments of the first set, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the first aspect of the invention provides a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a CKS scaffold to produce a chimeric nucleic acid encoding a chimeric protein of the first aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the first aspect provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a CKS scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein. In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fourth set of embodiments of the first aspect provides a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a CKS scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the first aspect provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub libraries comprising;

(i) a CKS scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second and third loops and the helical region of the CKS scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second and third loops and the helical region of the CKS scaffold.

A sixth set of embodiments of the first aspect provides a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a CKS scaffold,

(ii) a peptide ligand located in one of the first, second and third loops and the helical region of the CKS scaffold, and;

(iii) a peptide ligand located in another of the first, second and third loops and the helical region of the CKS scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the first aspect provides a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a CKS scaffold,

(ii) a peptide ligand located in one of the first, second and third loops and the helical region of the CKS scaffold, and;

(iii) a peptide ligand located in another of the first, second and third loops and the helical region of the CKS scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A second aspect of the invention is directed to chimeric proteins comprising coiled-coil scaffolds.

A first set of embodiments of the second aspect of the invention provide a chimeric protein comprising;

(i) a coiled-coil scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at one or more helices or loops of the coiled-coil scaffold.

A second set of embodiments of the second aspect of the invention provide a chimeric protein comprising;

i) a coiled-coil scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of any one of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the second aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a coiled-coil scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the second aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the second aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a coiled-coil scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said coiled-coil scaffold and said first and second peptide ligands,

wherein said peptide ligands are located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NO: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold; and expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fifth set of embodiments of the second aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a coiled-coil scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of S SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A sixth set of embodiments of the second aspect of the invention provide a library

comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a coiled-coil scaffold; and

(iii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein.

A seventh set of embodiments of the second aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a coiled-coil scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein at least one peptide ligand is diverse in said population, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

An eighth set of embodiments of the second aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a coiled-coil scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in the coiled- coil scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A third aspect of the invention is directed to chimeric proteins comprising Affibody scaffolds. A first set of embodiments of the third aspect of the invention provide a chimeric protein comprising;

(i) an Affibody scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at one or more helices and loops of the Affibody scaffold.

A second set of embodiments of the third aspect of the invention provide a chimeric protein comprising;

(i) an Affibody scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the third aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an Affibody scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the third aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the third aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an Affibody scaffold,

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said Affibody scaffold and said first and second peptide ligands, wherein said peptide ligands are located at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53 of the Affibody scaffold; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fifth set of embodiments of the third aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) an Affibody scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, and third helices and the first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein, wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A sixth set of embodiments of the third aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an Affibody scaffold; and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in two or more of the first, second, and third helices and the first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the of the Affibody scaffold of the chimeric protein, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in two or more of the first, second and third helices and the first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein.

A seventh set of embodiments of the third aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Affibody scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in two or more of the first, second, and third helices and first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein at least one peptide ligand is diverse in said population, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

An eighth set of embodiments of the third aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an Affibody scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 5) of the Affibody scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in the Affibody scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A fourth aspect of the invention is directed to chimeric proteins comprising trefoil scaffolds.

A first set of embodiments of the fourth aspect of the invention provide a chimeric protein comprising;

(i) a Trefoil scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at loops or b strands of the Trefoil scaffold.

A second set of embodiments of the fourth aspect of the invention provide a chimeric protein comprising; (i) a Trefoil scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the fourth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Trefoil scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the fourth aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the fourth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Trefoil scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said Trefoil scaffold and said first and second peptide ligands, wherein said peptide ligands are located at two or more loops (for example ,at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fifth set of embodiments of the fourth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a Trefoil scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A sixth set of embodiments of the fourth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Trefoil scaffold; and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein, and the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of SEQ ID NO: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein.

A seventh set of embodiments of the fourth aspect of the invention provide a method of producing a library of chimeric proteins comprising; (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Trefoil scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein,

wherein at least one peptide ligand is diverse in said population, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

An eighth set of embodiments of the fourth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Trefoil scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in the Trefoil scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A fifth aspect of the invention is directed to chimeric proteins comprising PDZ domain scaffolds.

A first set of embodiments of the fifth aspect of the invention provide a chimeric protein comprising;

(i) a PDZ scaffold, and

(ii) one or more, preferably two or more peptide ligands, said peptide ligands being located at two or more loops, a-helices or b-strands of the PDZ scaffold.

A second set of embodiments of the fifth aspect of the invention provide a chimeric protein comprising;

(i) a PDZ scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the fifth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an PDZ scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the fifth aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the fifth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an PDZ scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said PDZ scaffold and said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the SEQ ID NO: 79) of the PDZ scaffold; and

expressing the nucleic acid to produce said protein. In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fifth set of embodiments of the fifth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) an PDZ scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A sixth set of embodiments of the fifth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an PDZ scaffold; and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at wo or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein.

A seventh set of embodiments of the fifth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a PDZ scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein,

wherein at least one peptide ligand is diverse in said population, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins. An eighth set of embodiments of the fifth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an PDZ scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in the PDZ scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A sixth aspect of the invention is directed to chimeric proteins comprising ubiquitin or ubiquitin-like domain scaffolds

A first set of embodiments of the sixth aspect of the invention provide a chimeric protein comprising;

(i) an Ubiquitin scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at one or more helices and or loops of the Ubiquitin scaffold.

A second set of embodiments of the sixth aspect of the invention provide a chimeric protein comprising;

(i) an Ubiquitin scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; and between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305 and in structurally similar positions in SEQ ID NO: 293, 295, 297 and 299) of the Ubiquitin scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the sixth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an Ubiquitin scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the sixth aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the sixth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

a nucleic acid encoding an Ubiquitin scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said Ubiquitin scaffold and said first and second peptide ligands, wherein said peptide ligands are located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fifth set of embodiments of the sixth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) an Ubiquitin scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9;

between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A sixth set of embodiments of the sixth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Ubiquitin scaffold; and (ii) a peptide ligand comprising at least one diverse amino acid residue, wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein.

A seventh set of embodiments of the sixth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Ubiquitin scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein at least one peptide ligand is diverse in said population, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

An eighth set of embodiments of the sixth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an Ubiquitin scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein at least one amino acid residue in at least one peptide ligand in the Ubiquitin scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity. A seventh aspect of the invention is directed to chimeric proteins comprising GB1 scaffolds.

A first set of embodiments of the seventh aspect of the invention provide a chimeric protein comprising;

(i) a GB1 scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at loops or a- helices of the GB1 scaffold

A second set of embodiments of the seventh aspect of the invention provide a chimeric protein comprising;

(i) a GB1 scaffold, and

(ii) one or more, preferably two or more peptide ligands,

said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A third set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a GB1 scaffold, to produce a chimeric nucleic acid encoding a chimeric protein of the seventh aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A fourth set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

a nucleic acid encoding a GB1 scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said GB1 scaffold and said first and second peptide ligands, wherein said peptide ligands are located between residues 22 and 35 (preferably replacing residues 23, 24, 26, 27, 28 and 34-35 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) and between residues 46 and 49 of the GB1 scaffold; and expressing the nucleic acid to produce said protein.

A fifth set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

a nucleic acid encoding a GB1 scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said GB1 scaffold and said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A sixth set of embodiments of the seventh aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a GB1 scaffold; and

(ii) two or more peptide ligands, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in said library is diverse.

A seventh set of embodiments of the seventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a GB1 scaffold; and (ii) a peptide ligand comprising at least one diverse amino acid residue, wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein.

An eighth set of embodiments of the seventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a GB1 scaffold; and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located between at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein.

A ninth set of embodiments of the seventh aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a GB1 scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348 of the GB1 scaffold of the chimeric protein, wherein at least one peptide ligand is diverse in said population, and (b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

A tenth set of embodiments of the seventh aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a GB1 scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348 of the GB1 scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one peptide ligand in the GB1 scaffold of the chimeric proteins in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

An eighth aspect of the invention is directed to chimeric proteins comprising VWV scaffolds.

A first set of embodiments of the eighth aspect of the invention provide a chimeric protein comprising;

(i) a VWV scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or both of the first and second loops of the VWV scaffold of the chimeric protein.

The first loop may be at a position corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355, and residues 51 to 53 of SEQ ID NO: 359.

The second loop may be at a position corresponding to residues 23 to 25 of SEQ ID NO:

349 or SEQ ID NO: 357, residues 29 to 31 of SEQ ID NO: 351 , residues 32 to 34 of SEQ ID NO: 353, residues 24 to 26 of SEQ ID NO: 355 and residues 61 to 63 of SEQ ID NO: 359.

In some embodiments, the VWV scaffold may further comprise a helical region. Peptide ligands may be located in the helical region of the VWV scaffold. The helical region may be at a position corresponding to residues 14 to 32 of SEQ ID NOs: 412, 414 or 415 or residues In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the eighth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a VWV scaffold to produce a chimeric nucleic acid encoding a chimeric protein of the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the eighth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a VWV scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fourth set of embodiments of the eighth aspect of the invention provide a library

comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a VWV scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the eighth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a VWV scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the first loop (b) the second loop or (c) the helical region of the V V scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold.

A sixth set of embodiments of the eighth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a VWV scaffold,

(ii) a peptide ligand located in one of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the eighth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a VWV scaffold,

(ii) a peptide ligand located in one of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity. A ninth aspect of the invention is directed to chimeric proteins comprising Fibritin scaffolds.

A first set of embodiments of the ninth aspect of the invention provide a chimeric protein comprising;

(i) a Fibritin scaffold,

(ii) first peptide ligand in the disordered region of the Fibritin scaffold; and

(iii) a second peptide ligand in the coiled-coil subdomain of the Fibritin scaffold.

The coiled-coil subdomain may be at a position corresponding to residues 1 to 38 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 15 of SEQ ID NO 365; and the disordered region may be at a position corresponding to residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 16 to 27 of SEQ ID NO: 365.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the ninth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Fibritin scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the ninth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Fibritin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in the coiled-coil domain and the disordered region of the Fibritin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

In some preferred embodiments, one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.

A fourth set of embodiments of the ninth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a Fibritin scaffold, and

(ii) a first peptide ligand located in the coiled-coil subdomain of the Fibritin scaffold of the chimeric protein, and

(iii) a second peptide ligand located in the disordered region of the Fibritin scaffold of the chimeric protein,

wherein at least one amino acid residue in one of the first or second peptide ligand in said library is diverse.

A fifth set of embodiments of the ninth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Fibritin scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the disordered region and (b) the coiled-coil subdomain, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in the other of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold.

A sixth set of embodiments of the ninth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Fibritin scaffold,

(ii) a peptide ligand located in one of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold, and;

(iii) a peptide ligand located in another of (a) the disordered region and (b) the coiled- coil subdomain of the Fibritin scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins. A seventh set of embodiments of the ninth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Fibritin scaffold,

(ii) a peptide ligand located in one of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold, and;

(iii) a peptide ligand located in the other of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A tenth aspect of the invention is directed to chimeric proteins comprising aPP scaffolds.

A first set of embodiments of the tenth aspect of the invention provide a chimeric protein comprising;

(i) an avian pancreatic polypeptide (aPP) scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or both of the loop and the helical region of the aPP scaffold of the chimeric protein.

The loop may at a position in the aPP scaffold corresponding to residues 9 to 13 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415; and the helical region may at a position in the aPP scaffold corresponding to residues 14 to 32 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the tenth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding an aPP scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein. A third set of embodiments of the tenth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an aPP scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein said peptide ligands are located in (a) the loop and (b) the helical region of the aPP scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fourth set of embodiments of the tenth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) an aPP scaffold,

(ii) two peptide ligands, said peptide ligands being located in (a) the loop and (b) the helical region the aPP scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the tenth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an aPP scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold.

A sixth set of embodiments of the tenth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an aPP scaffold,

(ii) a peptide ligand located in one of (a) the loop and (b) the helical region of the aPP scaffold, and;

(iii) a peptide ligand located in the other of (a) the loop and (b) the helical region of the aPP scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the tenth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an aPP scaffold,

(ii) a peptide ligand located in one of (a) the first loop and (b) the helical region of the aPP scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop and (b) the helical region of the aPP scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

An eleventh aspect of the invention is directed to chimeric proteins comprising fibronectin scaffolds.

A first set of embodiments of the eleventh aspect of the invention provide a chimeric protein comprising;

(i) a fibronectin (FN3) scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein.

The first loop may be at a position in the FN3 scaffold corresponding to residues 14 to 15 of SEQ ID NO: 418 or SEQ ID NO: 420; the second loop may be at a position in the FN3 scaffold corresponding to residues 25 to 26 of SEQ ID NO: 418 or SEQ ID NO: 420; the third loop may be at a position in the FN3 scaffold corresponding to residues 43 to 44 of SEQ ID NO: 418 or SEQ ID NO: 420; and the fourth loop may be at a position in the FN3 scaffold corresponding to residues 81 to 82 of SEQ ID NO: 418 or SEQ ID NO: 420.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the eleventh aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a fibronectin scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the eleventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a fibronectin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is a member of a cellular degradation pathway, preferably a E3 ubiquitin ligase.

A fourth set of embodiments of the eleventh aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a fibronectin scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the eleventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a fibronectin scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the fibronectin scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the fibronectin scaffold.

A sixth set of embodiments of the eleventh aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a fibronectin scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the fibronectin scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the eleventh aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a fibronectin scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the fibronectin scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity. A twelfth aspect of the invention is directed to chimeric proteins comprising Zn finger scaffolds.

A first set of embodiments of the twelfth aspect of the invention provide a chimeric protein comprising;

(i) a Zn finger scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or more of the first loop, second loop, and helical region of the Zn finger scaffold of the chimeric protein.

The first loop may be at a position in the Zn finger scaffold corresponding to residues 6 to 9 of SEQ ID NO: 423; the second loop may be at a position in the Zn finger scaffold corresponding to residues 11 to 12 of SEQ ID NO: 423 and the helical region may be at a position in the Zn finger scaffold corresponding to 17 to 28 of SEQ ID NO: 423.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the twelfth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a Zn finger scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the twelfth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Zn finger scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.

A fourth set of embodiments of the twelfth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a Zn finger scaffold,

(ii) two or more peptide ligands, wherein a first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and a second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the twelfth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Zn finger scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first loop, second loop and helical region of the Zn finger scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first loop, second loop and helical region of the Zn finger scaffold.

A sixth set of embodiments of the twelfth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Zn finger scaffold,

(ii) a first peptide ligand located in one of the first loop, second loop and helical region of the Zn finger scaffold, and;

(iii) a second peptide ligand located in another of the first loop, second loop and helical region of the Zn finger scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the twelfth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Zn finger scaffold,

(ii) a first peptide ligand located in one of the first loop, second loop and helical region of the Zn finger scaffold, and;

(iii) a second peptide ligand located in another of the first loop, second loop and helical region of the Zn finger scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

A thirteenth aspect of the invention is directed to chimeric proteins comprising SH3 scaffolds.

A first set of embodiments of the thirteenth aspect of the invention provide a chimeric protein comprising;

(i) an SH3 scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein.

The first loop may be at a position in the SH3 scaffold corresponding to residues 9 to 24 of SEQ ID NO: 653; residues 24 to 39 of SEQ ID NO: 655; or 13 to 21 of SEQ ID NO: 657; the second loop may be at a position in the SH3 scaffold corresponding to residues 31 to 35 of SEQ ID NO: 653; residues 45 to 56 of SEQ ID NO: 655; or residues 32 to 41 of SEQ ID NO: 657; the third loop may be at a position in the SH3 scaffold corresponding to residues 44 to 46 of SEQ ID NO: 653 residues 62 to 63 of SEQ ID NO: 655; or residues 53 to 62 of SEQ ID NO: 657; and the fourth loop may be at a position in the SH3 scaffold corresponding to residues 55 to 56 of SEQ ID NO: 653; residues 69 to 71 of SEQ ID NO: 655; or residues 68 to 70 of SEQ ID NO: 657.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase. A second set of embodiments of the thirteenth aspect of the invention provide a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an SH3 scaffold to produce a chimeric nucleic acid encoding a chimeric protein of the invention; and

expressing said chimeric nucleic acid to produce the chimeric protein.

A third set of embodiments of the thirteenth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an SH3 scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some preferred embodiments, one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.

A fourth set of embodiments of the thirteenth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) an SH3 scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the thirteenth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an SH3 scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the SH3 scaffold, and the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the SH3 scaffold.

A sixth set of embodiments of the thirteenth aspect of the invention provide a method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an SH3 scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the SH3 scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the SH3 scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the thirteenth aspect of the invention provide a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an SH3 scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

A fourteenth aspect of the invention is directed to chimeric proteins comprising a cystine knot (CK) scaffold (also called a cyclotide scaffold). A first set of embodiments of the fourteenth aspect of the invention provide a chimeric protein comprising;

(i) a cystine knot scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or more, preferably two or more, of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein.

The first loop may be at a position in the CK scaffold corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842, residues 2 to 7 of SEQ ID NO: 844; residues 3 to 8 of SEQ ID NO: 846 and residues 3 to 4 of SEQ ID NO: 848; the second loop may be at a position in the CK scaffold corresponding to residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 3, residues 9 to 13 of SEQ ID NO: 844; residues 10 to 14 of SEQ ID NO: 846 and residues 6 to 15 of SEQ ID NO: 848; the third loop may be at a position in the CK scaffold corresponding to residues 11 to 14 of SEQ ID NO: 840; residues 11 to 16 of SEQ ID NO: 3, residues 15 to 17 of SEQ ID NO: 844; residues 16 to 18 of SEQ ID NO: 846 and residues 17 to 19 of SEQ ID NO: 848; the fourth loop i may be at a position in the CK scaffold corresponding to residue 16 of SEQ ID NO: 840; residue 18 of SEQ ID NO: 3, residue 19 of SEQ ID NO: 844; residue 20 of SEQ ID NO: 846 and residues 21 to 27 of SEQ ID NO: 848; the fifth loop may be at a position in the CK scaffold corresponding to residues 18 to 21 of SEQ ID NO: 840; residues 20 to 23 of SEQ ID NO: 3, residues 21 to 25 of SEQ ID NO: 844; residues 22 to 26 of SEQ ID NO: 846 and residues 29 to 32 of SEQ ID NO: 848; and the sixth loop may be at a position in the CK scaffold corresponding to residues 23 to 30 of SEQ ID NO: 840; residues 25 to 31 of SEQ ID NO: 3, residues 27 to 35 of SEQ ID NO: 844; residues 28 to 30 of SEQ ID NO: 846 and residue 34 of SEQ ID NO: 848.

In some preferred embodiments of the first set, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase.

A second set of embodiments of the fourteenth aspect of the invention provides a method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a cystine knot scaffold to produce a chimeric nucleic acid encoding a chimeric protein of the first aspect; and

expressing said chimeric nucleic acid to produce the chimeric protein. A third set of embodiments of the fourteenth aspect provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a cystine knot scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

A fourth set of embodiments of the fourteenth aspect provides a library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a cystine knot scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

A fifth set of embodiments of the fourteenth aspect provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub libraries comprising;

(i) a cystine knot scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third, fourth, fifth and sixth loops of the CKS scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third, fourth, fifth and sixth loops of the CKS scaffold.

A sixth set of embodiments of the fourteenth aspect provides a method of producing a library of chimeric proteins comprising; (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a cystine knot scaffold,

(ii) a peptide ligand located in one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold, and;

(iii) a peptide ligand located in another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

A seventh set of embodiments of the fourteenth aspect provides a method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a cystine knot scaffold,

(ii) a peptide ligand located in one of the first, second and third loops and the helical region of the cystine knot scaffold, and;

(iii) a peptide ligand located in another of the first, second and third loops and the helical region of the cystine knot scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

Other aspects and embodiments of the invention are described in more detail below.

Brief Description of Fiaures

Figure 1 shows a schematic representation of a library of degradation-inducing chimeric proteins. The library shown is for use in targeting b-catenin for degradation. These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway. Each of the target-binding peptides is derived from a different protein that interacts with b-catenin (see Table 1). Each of the degradation pathway-binding peptides (referred to as“degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways);‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in Table 2. The schematic illustrates the combinatorial“plug-and-play” nature of these libraries, in terms of the ability to slot in any target-recruiting peptide ligand and degradation-pathway-recruiting peptide ligand. The other factor that can be varied in the library arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery. Once the library is constructed, it can then be screened in cell-based assays in order to identify the best combination of two peptide ligands and their positions within the scaffold that induces the greatest reduction in target protein levels. The same panel of diverse degradation pathway components can be used for screen for degradation of any target.

Figure 2 shows a schematic of the structure of the CKS1 scaffold (2A) and modelled structures of bifunctional CKS1 molecules (2B and C). Figure 2A shows a model of the CKS1 scaffold with sites for peptide grafting and the natural binding site for Skp2 (the substrate-recognition subunit of the E3 ubiquitin ligase SCF ^Skp2 ) highlighted and annotated. Figure 2B shows a model of a bifunctional CKS1 molecule with a peptide targeting B- catenin-/ KRAS grafted onto a loop (site 1) and the natural binding site for Skp2 indicated. Figure 2C shows a model of a bifunctional CKS1 molecule with a peptide targeting B-catenin / KRAS grafted onto a loop (site 2) and the natural Skp2-binding site indicated. Highlighted in black are the loops or helices targeting B-catenin / KRAS and the Skp2-binding site. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 1 DKS as template. Images were generated using PyMol.

Figure 3 shows the modelled structure of the complex of Skp2 (the substrate-recognition subunit of the E3 ubiquitin ligase SCF ^Skp2 ) and the Cks1 scaffold with a grafted loop.

Figure 4 shows a helical wheel model of SEQ ID NO: 1 residues 40 to 45 where X represents substitutions to the helix on the solvent accessible face to create a new ligand binding surface.

Figure 5A shows b-catenin degradation using bispecific CKS1 constructs as measured using a HiBiT lytic assay (UNT: Untreated, Scr: Scrambled siRNA, LIPO: Lipofectamine only siRNA: b-catenin-targeted siRNA). Figure 5B shows KRAS degradation using bispecific CKS1 constructs as measured using a HiBiT lytic assay. Figure 5C shows a schematic representation of components used to build the CKS1 constructs. CKS1 : CKS1 protein, which acts as a substrate adaptor for the E3 ligase SCF ^Skp2 . Residues involved in binding to the protein Cdk2 and to the protein p27 have been mutated. HA: HA tag; Phospho: a beta- catenin binding sequence from the protein ARC (Adenomatous polyposis coli); SOX: a beta- catenin binding sequence from the protein SOX; KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605- 61 1). RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 1 1 : 517-522); aFT: a KRAS-binding sequence from the protein alpha farnesyl transferase; FT: a KRAS-binding sequence from the protein alpha farnesyl transferase; Skp2-binding: indicates residues from native CKS1 required for binding to the E3 sCF ^Skp2 ; Skp2-, cdk2- and p27-binding: this construct has the residues from native CKS1 required for binding to the E3 SCF ^Skp2 and also for binding to cdk2 and p27. The amino acid sequences of the constructs shown in Figure 5C are set out in Table 4.

Figure 6 shows the quantification of expression of mono- and bispecific CKS1 constructs in the cell line MIA PaCa-2 24 hours after transfection (Scram: Scrambled siRNA, siRNA: b- catenin targeted siRNA).

Figure 7 shows a schematic of the structure of a representative coiled-coil scaffold derived from the PDB structure.

Figure 8A shows a schematic of the coiled-coil scaffold, based on PDB 1 cxz, with examples of sites at which binding peptides can be grafted highlighted. Figure 8B and 8C show models of bifunctional coiled-coil molecules with peptides for binding to B-catenin/ KRAS and to an E3 ligase grafted onto alpha-helices. Figure 8D shows a model of a bifunctional coiled-coil molecule with peptides for binding to B-catenin-/ KRAS-binding and to an E3 ligase grafted onto a loop and a helix, respectively, highlighted. B-catenin Distances between the two targeting peptides are shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 1CXZ as template. Images were generated using PyMol.

Figure 9 shows a model of coiled-coil scaffold-mediated beta-eaten in ubiquitination through the MDM2 ubiquitin ligase. Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between MDM2, the coiled-coil scaffold and the beta-eaten in is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the p53 degron peptide (sequence FAAYWNLLSAYG) bound to the N- terminal domain of MDM2.

Figure 10 shows b-catenin degradation using bispecific coiled-coil constructs as measured using a HiBiT lytic assay (Figures 10A and 10B). Figure 10C shows a schematic representation of components used to build the coiled-coil (CC) construct. CC: CC is a coiled-coil from the RHOA-binding effector domain of the protein kinase PKN/PRK1 (PDB 1CXZ). All of the lysines, one of which is functionally important, and two other residues required for the native function, have been substituted; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli): AXIN: an alpha- helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta- catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al. , 2010, ChemBioChem 11 : 517- 522); p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2; Puc: a degron sequence from the protein Puc that binds the E3 Cul3-SPOP; NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1 ; PHYL: a degron sequence from the protein PHYL that binds the E3 SI AH; Trib: a degron sequence from the protein Trib that binds the E3 COP1 ; PAM2: a degron sequence from the protein PAM2 that binds the E3 UBR5; CDC25B: a degron sequence from the protein CDC25B that binds the E3 beta- TRCP; p53: an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2. The amino acid sequences of the constructs shown in Figure 10C are set out in Table 12.

Figure 11 A and 1 1 B show the quantification of expression of bispecific coiled-coil constructs in the cell line MIA PaCa-2 24 hours after transfection. Figure 1 1C shows a western blot of b-catenin using HEK293 transfected with bifunctional constructs or controls. Total protein staining SDS-PAGE of HEK293 cell extract 24hrs (top left panel) and 48hrs (top right panel) after transfection with bifunctional constructs or controls is shown. Total b-catenin western blot of HEK293 cell extract 24hrs (bottom left panel) and 48hrs (bottom right panel) after transfection with bifunctional constructs or controls is also shown, along with densitometric analysis of total b-catenin Western Blot showing total area (Figure 1 1 D top panel) or normalised values (Figure 11 D bottom panel).

Figure 12 shows a schematic of the structure of a representative Affibody scaffold with a grafted loop 1 (between H1 and H2 helices) and a grafted H3 helix. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motif was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.

Figure 13 shows a CLUSTAL W alignment of representative sequences of Affibody domains Figure 14A shows a schematic of the modelled structures of a bifunctional Affibody molecule with binding peptides for B-catenin and for an E3 ligase grafted onto a helix and a loop, respectively. Figure 14B shows a model of a bifunctional Affibody molecule, with peptides for binding to B-catenin and to an E3 ligase grafted onto two helices, in complex with B-catenin and the N-terminal domain of MDM2.

Figure 15 shows a model of Affibody scaffold-mediated KRAS ubiquitination via the Cul3- Keapl E3 ubiquitin ligase complex. The E3 Cul3-Keap1-E2 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107). The Affibody scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl . The geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl . Two different views are shown.

Figure 16A shows b-catenin degradation using bispecific Affibody constructs as measured using a HiBiT lytic assay (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 16B shows a schematic representation of components used to build the Affibody constructs. Affibody: A protein scaffold based on the Z domain of a Staphylococcus aureus protein A (PMID: 18435759); HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha- helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 16.

Figure 17 shows the quantification of expression of mono- and bispecific Affibody constructs in the cell line MIA PaCa-2 24 hours after transfection. Scram: Scrambled siRNA, siRNA: b- catenin targeted siRNA.

Figure 18 shows a schematic of the structure of a representative Trefoil scaffold derived from PDB structure code 2AFG.

Figure 19 shows the secondary structure of a representative Trefoil scaffold. The beta- strands represented as arrows and italic text, helices as rectangles and bold text.

Unstructured regions connect the secondary structure elements. In some examples of Trefoil domain, helices are present as a part of the long beta-turns. Figure 20A shows a schematic of the structure of the Trefoil scaffold with examples of sites for peptide grafting highlighted in black and annotated. Figure 20B shows a model of a bifunctional Trefoil molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto loops. Highlighted in black are loops for binding to b-catenin / KRAS and to an E3 ligase. The distance between the two targeting peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 3PG0 as template. Images were generated using PyMol.

Figure 21 A shows b-catenin degradation determined using a HiBiT lytic assay for Trefoil constructs. Figure 21 B shows KRAS degradation determined using a HiBiT lytic assay for Trefoil constructs. Figure 21 C show a schematic representation of components used to build the Trefoil constructs. Trefoil: The Trefoil scaffold based on a designed 3-fold symmetric non-functional protein (PMID: 22178248, pdb code 3PG0) with lysines removed based on known substitutions at these positions in an NCBI alignment of the protein family, from which the designed sequence is derived; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al. ,

Biochem. Biophys. Res. Commun. 2017 484: 605-611); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 20.

Figure 22 shows a quantification of the expression of single and bifunctional Trefoil constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA. Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA.

Figure 23 shows a schematic of the structure of a representative PDZ scaffold.

Figure 24 shows a schematic of the structure of the PDZ scaffold (24A) and the modelled structure of a bifunctional PDZ protein (24 B). Figure 24A shows a model of the PDZ scaffold with examples of sites for peptide grafting highlighted in black and annotated. Figure 24B shows a model of the bifunctional PDZ molecule with peptides highlighted in black for binding to B-catenin / KRAS and to an E3 ligase grafted onto a helix and a loop respectively. The distance between the two targeting peptides is shown. All models were generated using SWSS-MODEL (expasy Webserver) with pdb 3JXT as template. Images were generated using PyMol. Figure 25 shows a model of PDZ scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, PDZ scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (from the protein Nrf2) (sequence ETGE) bound to the b-propeller domain of Keapl .

Figure 26A shows b-catenin degradation determined using a HiBiT lytic assay for PDZ constructs (UNT: Untreated SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 26B shows KRAS degradation determined using a HiBiT lytic assay for PDZ constructs (UNT: Untreated, SRC: Scrambled siRNA, LIPO:

Lipofectamine only, siRNA: KRAS targeted siRNA). Figure 26C shows a schematic representation of components used to build the PDZ constructs; PDZ: PSD95 PDZ3 domain was used as a scaffold (PMID: 15820976) with lysine residues substituted. PDZ domains have low affinity for their natural ligands, as they participate in multivalent interactions, and therefore the isolated domain will not be functional; Phospho: a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha-helical beta- catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9. SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 25.

Figure 27 shows a quantification of the expression of single and bifunctional PDZ constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA).

Figure 28 shows a schematic of the structure of a representative Ubiquitin scaffold derived from PDB structure code 2KOX.

Figure 29 shows a schematic of the modelled structure of a bifunctional ubiquitin molecule with binding peptides for B-catenin and for an E3 ligase grafted onto loops. All models were generated using SWISS-MODEL (expasy Webserver). Images were generated using PyMol. Figure 30 shows a model of Ubiquitin scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with

MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, Ubiquitin scaffold and the Cul3- Keapl E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl Two different views are shown

Figure 31A shows b-catenin degradation determined using a HiBiT lytic assay for ubiquitin constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 31 B shows a schematic representation of components used to build the Ub (ubiquitin)/ UBL (Ubiquitin-like) constructs; hPLIC2: A ubiquitin-like domain that binds to the Rpn13 subunit of the proteasome; Raf-RBD: RAS-binding domain of the protein C-Raf (also known as Raf-1). Raf-RBD has a Ubiquitin structure; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-eaten in binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha- helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010,

ChemBioChem 11 : 517-522); KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611); For PPX107, PPX108, PPX110 and PPX11 , the residues involved in RAS-binding function of Raf-RBD have been replaced with the RAS-binding motif RBP or KBL; p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2; NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1 ; KRAS-binding: indicates that the PPX sequence contains residues of Raf-RBD required for binding to KRAS; Proteasome-binding: indicates that the PPX sequence contains residues of hPLIC2 required for binding to the proteasome. The amino acid sequences of these constructs are shown in Table 29.

Figure 32 shows a quantification of the expression of single and bifunctional ubiquitin constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA. (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo:

Lipofectamine only) Figure 33 shows a schematic of the structure of representative GB1 domain with examples of sites for peptide grafting annotated.

Figure 34 shows a schematic of the modelled structure of a bifunctional GB1 molecule with peptides binding to B-catenin / KRAS and to an E3 ligase grafted onto loops. The distance between the two targeting peptides is shown. AH models were generated using SWISS- MODEL (expasy Webserver) with pdb 1GB4 as template. Images were generated using

PyMol.

Figure 35 shows two different views of the modelled structure of the complex of loop-helix- grafted GB1 domain (in grey) in complex with KRAS and the E2-E3 Cullin-Keapl . The figure shows a model of GB1 scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al (Free Rad Biol & Med (2015) 88 101-107). The GB1 scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .The geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl Two different views are shown). Grafting of loop motifs was modelled with

MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, GB1 scaffold and the Cul3- Keapl E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl

Figure 36A shows b-catenin degradation determined using a HiBiT lytic assay for GB1 constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 36B shows a schematic representation of components used to build the GB1 constructs. GB1 : Stabilised version of the B1 IgG binding domain of protein G from Streptococcus (PMID: 9628485) with the lysine residues substituted. Lysine substitutions and loop insertions will disrupt the native function; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha- helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS- binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2. NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 33.

Figure 37 shows a quantification of the expression of single and bifunctional GB1 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).

Figure 38 shows a schematic representation of the structure of the VWV domain of PIN1.

Figure 39 shows modelled structures of a VWV scaffold with two grafted loops and with a grafted helix and a grafted loop. Grafting of the loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera.

Figure 40 shows a schematic of structure of the WW scaffold and a modelled structure of the V V scaffold with two grafted peptides. Figure 40 shows the WW scaffold with examples of sites for peptide grafting highlighted and annotated. Figure 40 shows a model of the WW scaffold with binding peptides for B-catenin / KRAS and for an E3 ligase grafted onto loops highlighted. B-cateninThe distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 2M8I as template. Images were generated using PyMol.

Figure 41 shows a helical wheel of the helixWW scaffold of SEQ ID NO: 11. The Xs represent the isomorphic replacement of amino acids to form a binding interface on the solvent-accessible side of the helix.

Figure 42A shows b-catenin degradation determined using a HiBiT lytic assay for WW constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LI PO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 42 B shows a schematic representation of components used to build the WW constructs. WW: a thermodynamically stabilised version of the WW domain from PIN1 (PMID: 23378640) with the lysine residue substituted. Sequences are placed into a loop used to bind proline- rich peptide ligands. WW domains participate in multivalent interactions in nature and have low affinity for their natural ligands, so the isolated domain is not expected be functional; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517- 522); KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611). NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 38.

Figure 43 shows a quantification of the expression of single and bifunctional VWV constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, Lipo: Lipofectamine only, siRNA: b-catenin targeted siRNA).

Figure 44 shows the modelled structure of the longer (44A) and shorter (44 B) Fibritin domains with a grafted loop. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.

Figure 45A shows a schematic of the modelled structure of a Fibritin domain with a grafted loop and a grafted helix. Figures 45B-D show schematics of the modelled structures of a single chain of a Fibritin domain (pdb 1aa0) with binding peptides for B-catenin / KRAS and for an E3 ligase grafted onto different helices and loops. Distances between the two binding peptides are shown.

Figure 46 shows a model of Fibritin scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107). The Fibritin scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .The geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl . Two different views are shown.

Figure 47A shows b-catenin degradation determined using a HiBiT lytic assay for Fibritin constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 47B shows a schematic representation of components used to build the Fibritin constructs. Fibritin: The trimerization domain of T4 phage Fibritin with the lysines replaced; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 41.

Figure 48 shows a quantification of the expression of single and bifunctional Fibritin constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.

Figure 49 shows schematics of the structures of aPP scaffold (49A) and PPa scaffold (49C) and the modelled structures of their bifunctional molecules (49 B and 49D). Figure 49A shows the aPP scaffold with examples of sites for peptide grafting highlighted in black and annotated. Figure 49 B shows a model of a bifunctional aPP molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto a loop and a helix, respectively, highlighted in black. Figure 49C shows a model of PPa scaffold with sites for peptide grafting highlighted in black and annotated. Figure 49D shows a model of a bifunctional PPa molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto a helix and a loop, respectively, highlighted in black. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 2BF9 (for aPP constructs) and with pdb 5L02 as template. Images were generated using PyMol.

Figure 50 shows b-catenin degradation determined using a HiBiT lytic assay for aPP constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).

Figure 51 shows a schematic representation of components used to build the aPP and PPa constructs. aPP: aPP is an avian pancreatic polypeptide domain from a peptide hormone. Grafting of degron and target-binding sequences will abolish its native function. The native sequence contains no lysine residues; HA: HA tag; PPa: PPa is a designed (non-functional) aPP protein in which the sequence optimised for stability (PMID 31251570). Lysine residues in the original sequence have been replaced with arginine residues; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha- helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta- catenin binding sequence from the protein BCL9; RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); KBL: a KRAS- binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-61 1); p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2; NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1 ; WNK4: a degron sequence from the protein WNK4 that binds the E3 KLHL2; p53: an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2. The amino acid sequences of these constructs are shown in Table 45.

Figure 52 shows schematics of the structure of the Fibronectin type III scaffold (52A) and the modelled structure of a bifunctional Fibronectin type III molecule (52 B). Figure 52A shows a Fibronectin type III scaffold with examples of sites for peptide grafting highlighted and annotated. Figure 52 B shows a model of a bifunctional Fibronectin type III molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto loops, highlighted in black. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 4U3H as template. Images were generated using PyMol.

Figure 53A shows b-catenin degradation determined using a HiBiT lytic assay for FN3 constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 53B shows a schematic representation of components used to build the Fibronectin type III (FN3) constructs. FN3: A non-functional stabilised Fibronectin type III consensus sequence based on the alignment of multiple FN3 domains (PMID:25691761) with the single lysine replaced with an arginine; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 48.

Figure 54 shows a quantification of the expression of single and bifunctional FN3 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).

Figure 55 shows a modelled structure of a Zinc finger domain with a grafted loop and a grafted helix. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motifs was modelled with

MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.

Figure 56 shows a model of Zn finger scaffold-mediated KRAS ubiquitination via the Cul3- Keapl E3 ubiquitin ligase complex. The E3 Cul3-Keap1-E2 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107). The Zn finger scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .The geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl

Figure 57 A shows b-catenin degradation determined using a HiBiT lytic assay for ZF constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 57B shows a schematic representation of components used to build the Zinc-finger (ZF) constructs. ZF: based on the second C2H2 type Zinc-finger domain of ZNF32. Lysines are substituted, which abolishes the natural DNA binding function; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); KBL: a KRAS-binding sequence identified by phage display

(Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 51.

Figure 58 shows a quantification of the expression of single and bifunctional ZF constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).

Figure 59 shows a modelled example of a bifunctional SH3 scaffold created by grafting first and second peptide ligands onto the first and second loops (R-loop and Src loop, respectively). Grafting of the loop peptides was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.

Figure 60 shows the SH3 domain from the T cell adapter protein ADAP that has an N- terminal helix (referred to here as hSH3) (pdb 1 R 19) , with examples of sites for peptide grafting highlighted and annotated. Images were generated using PyMol.

Figure 61 shows a modelled example of an SH3 scaffold bound to its natural ligand, namely C-Myc, with a degron peptide grafted onto the third loop.

Figure 62 shows b-catenin degradation determined using a HiBiT lytic assay for src

Homology-3 (SH3) constructs in two separate assays (Figures 62A and 62 B) (UNT:

Untreated cells, SCRAM/Scr: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA). Figure 62C shows a schematic representation of components used to build the src Homology-3 (SH3) constructs. Fyn-SH3: the SH3 domain of Fyn (a Src family tyrosine kinase). Degrons have been inserted into n-src loop, thereby abolishing its native function. Lysine residues in the native sequence have been replaced with arginine residues. hSH3: the SH3 domain from the T cell adapter protein ADAP that has an N-terminal helix. All helical binding sequences are grafted onto this helix. The grafting process, together with the substitution of several lysine residues, knocks out the native lipid-binding activity of this protein. Other lysine residues in the native sequence have been replaced with arginine residues. Phospho: a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli). AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN. BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9.

SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1). KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al. , Biochem. Biophys. Res. Commun. 2017 484: 605-611). P27: a degron sequence from the protein p27 that binds the E3 SCFSkp2. Puc: a degron sequence from the protein Puc that binds the E3 Cul3-SPOP. NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. PHYL: a degron sequence from the protein PHYL that binds the E3 SIAH. Trib: a degron sequence from the protein Trib that binds the E3 COP1. PAM2: a degron sequence from the protein PAM2 that binds the E3 UBR5. CDC25B: a degron sequence from the protein CDC25B that binds the E3 beta-TRCP. p53: an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2. The amino acid sequences of the constructs are shown in Table 55.

Figure 63 shows Western blot of B-catenin using HEK293 transfected with bifunctional SH3 construct or controls. Total protein staining SDS-PAGE of HEK293 cell extract 24hrs (top left panel) and 48hrs (top right panel) after transfection with bifunctional constructs or untransfected controls. Total B-catenin western blot of HEK293 cell extract 24hrs (bottom left panel) and 48hrs (bottom right panel) after transfection with bifunctional SH3 construct or untransfected controls. Densitometric analysis of total b-catenin Western Blot showing total area (Figure 63 CONTD top panel) or normalised values (Figure 63 CONTD bottom panel).

Figure 64 shows a quantification of the expression of single and bifunctional SH3 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA. Scram: Scrambled sRNA, siRNA: B-catenin targeted siRNA, Lipo: Lipofectamine only.

Figure 65 shows isothermal titration calorimetry (ITC) data of grafted cystine knot scaffold (PPX259) binding to Keapl in the presence of reducing agent (0.3 mM TCEP) Figure 66 shows ITC data of grafted cystine knot scaffold (PPX259) binding to Keapl in very low concentration of reducing agent (0.015 mM TCEP)

Figure 67 shows competition fluorescence polarisation data for grafted cystine knot scaffolds (PPX252 and PPX253) binding to MDM2.

Figure 68 shows a schematic representation of components used to build the cystine knot constructs. Cyclotide: Cyclotide is a disulphide-rich peptide sequence based on the cyclotide MCoTI-ll (Momordica cochinchinensis trypsin inhibitor-ll) (Felizmenio-Quimio, M. E. et al., J Biol. Chem. 2001 276: 22875-22882). Under oxidising conditions, disulphide bonds form, which creates loops onto which the target-binding or E3 ligase-engaging peptides can be grafted. PPX250, PPX251 , PPX252, PPX253, PPX254, PPX255 and PPX256 are N-to-C cyclised, thereby creating an additional loop for peptide grafting. Biotin: Biotin tag. Phospho: a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli). AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN. BCL9: an alpha- helical beta-catenin binding sequence from the protein BCL9. SOS: an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1). KBL: a KRAS- binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611). Abltide: A peptide that binds to BCR-ABL. p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2. NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. Trib: a degron sequence from the protein Trib that binds the E3 COP1. p53: an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2. KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611). RBP: a KRAS- binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522).

Figure 69 shows a schematic of the modelled structure of a cyclotide with two grafted loops, Loop 2 and Loop 6.

Detailed Description

This invention relates to chimeric proteins that comprise a monomeric peptide scaffold (i.e. grafted peptide scaffolds). One or more peptide ligands are located in the scaffold of a chimeric protein. The peptide ligands may be to the same or different target molecules and the chimeric proteins of the first to fourteenth aspects may be multi-functional and/or multi valent. Chimeric proteins as described herein may be useful in a range of therapeutic and diagnostic applications. A scaffold is a protein with stable secondary and tertiary structures that tolerates the insertion or grafting of one or more heterologous peptide ligands into the protein sequence i.e. the scaffold retains its structure in the presence of inserted/grafted peptide ligand(s). The ability of a heterologous peptide ligand to bind to target molecules is retained when the heterologous peptide ligand is located within the scaffold, and the scaffold may serve to constrain the heterologous peptide ligand in a binding-competent conformation. A scaffold in which one or more heterologous peptide ligands have been inserted may be referred to herein as a grafted scaffold or a chimeric protein.

A variant of a reference chimeric protein, construct, scaffold or peptide ligand sequence set out herein may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3,

4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith- Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147. 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequence described herein.

For example, a scaffold of the first to the fourteenth aspects may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands. For example, aromatic residues in the scaffold may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of scaffold sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.

In some embodiments, lysine residues in a scaffold of the first to the fourteenth aspects may be replaced by a different residue, such as arginine to prevent unwanted ubiquitination and subsequent degradation. This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC).

(i) CKS Scaffolds

A first aspect relates to chimeric proteins that comprise CKS scaffolds (i.e. grafted CKS scaffolds). One or more peptide ligands are located in the CKS scaffold of the chimeric protein, for example in the first, second or third loops or the helical region.

The CKS1 (Cyclin-dependent kinases regulatory subunit 1) domain and CKS2 (Cycl in dependent kinases regulatory subunit 2) domain are well known and well-characterized in the prior art. CKS1 acts as a substrate adaptor for the E3 ubiquitin ligase SCF ^Sk P ² by binding both SCF ^Sk P ² and a subset of substrates resulting in ubiquitination of those substrates. One such substrate is the cell-cycle inhibitor p27), and CKS proteins have a binding site for cyclin-dependent kinase 2 (CDK2) that further enhances the efficiency of p27 ubiquitination by SCF ^Skp2 .

The structure of the CKS domain is well known in the art (see for example PFAM 01 11).

The invention adopts the well understood sequence-structure relationships of CKS domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules. The ability of human CKS protein CKS1 to bind three different proteins simultaneously, combined with its natural function as an E3-substrate adaptor, makes it especially suitable for use in the invention described.

As used herein, a CKS scaffold 4-stranded b-sheet protein with a short alpha-helix (i.e. a CKS domain structure).

A CKS scaffold, as used herein, has a length of 60 to 80 amino acids, preferably about 70 amino acids or 69 amino acids.

A CKS scaffold may have a MW of about 7600 Da. Suitable CKS scaffolds useful according to the invention include Human CKS protein Cks1 (Uniprot P61024; Gene ID 1163; NP_001817.1) and Human CKS protein Cks2 (Uniprot P33552) or variants of either of these. The sequences of Cks1 and Cks2 are shown in Table 3.

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable CKS scaffolds useful according to the invention without any limitation: 2ASS, 2AST, 4YC3, 1 DKS and 4YC6.

Suitable CKS scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44: D279-D285).

In some embodiments, a CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

The CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 2 or a variant thereof.

In some embodiments, a CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 3 or a variant thereof.

The CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 4 or a variant thereof.

Preferred CKS scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in a CKS domain may be replaced by either a polar or charged amino acid, preferably Arg or Glu, to generate a CKS scaffold. A suitable lysine-free CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 5 or a variant thereof.

(SEQ ID NO: 5; replacements of Lys residues underlined)

The lysine-free CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 6 or a variant thereof.

The first loop of the CKS scaffold is located within the position corresponding to residues 25 to 39 of SEQ ID NO: 1 and SEQ ID NO: 665 and residues 31 to 39 of SEQ ID NO: 3.

The second loop of the CKS scaffold is located within the position corresponding to residues 46 to 54 of SEQ ID NO: 1 and SEQ ID NO: 665, and residues 46 to 53 of SEQ ID NO: 3.

The third loop of the CKS scaffold is located within the position corresponding to residues 58 to 64 of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 665.

The helix of the CKS scaffold is located within the position corresponding to 40 to 45 of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 665.

In some embodiments, a CKS scaffold may display binding activity. For example, a CKS scaffold may bind to the E3 ubiquitin ligase SCF ^Skp2 , in the absence of an inserted peptide ligand. Skp2 binding may be mediated by the helical region corresponding to residues 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3.

A grafted CKS scaffold that binds to Skp2 may further comprise a peptide ligand in a loop of the CKS scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). The peptide ligand may be heterologous.

In some embodiments, a grafted CKS scaffold may contain a peptide ligand within the first loop (a“loop peptide”) i.e. the loop corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3. In some preferred embodiments, a peptide ligand may be located between residues corresponding to 25 and 28 of SEQ ID NO: 1 and 31 to 34 of SEQ ID NO: 3. For example, a peptide ligand may be located in the CKS scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37 or 38 of SEQ ID NO: 1. The peptide ligand may be inserted into the CKS scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 26, 27, 28, 29, 30, 31 ,

32, 33, 34, 35, 36, 37, 38, 39 or 40 of SEQ ID NO: 1. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 25 to 39 of SEQ ID NO: 1.

In some embodiments, a grafted CKS scaffold may contain a peptide ligand within the second loop (a“loop peptide”) i.e. the loop corresponding 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3. For example, a peptide ligand may be located in the CKS scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 45, 46, 47, 48, 49, 50, 51 , 52, or 53 of SEQ ID NO: 1. The peptide ligand may be inserted into the CKS scaffold

immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 47, 48, 49, 50, 51 , 52, 53, 54, or 55 of SEQ ID NO: 1. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the second loop. For example, a peptide ligand may replace the loop residues corresponding 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3.

In some embodiments, a grafted CKS scaffold may contain a peptide ligand within the third loop (a“loop peptide”) i.e. the loop corresponding to residues 58 to 64 of SEQ ID NO: 1 and SEQ ID NO: 3. In some preferred embodiments, a peptide ligand may be located between residues 61 and 64 of SEQ ID NO: 1 and 3. For example, a peptide ligand may be located in the CKS scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 57, 58, 59, 60, 61 , 62, or 63 of SEQ ID NO: 1 or 3. The peptide ligand may be inserted into the CKS scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 59, 60, 61 , 62, 63, 64, or 65 of SEQ ID NO: 1. The peptide ligand may be added to the third loop or may replace one or more residues of the third loop. For example, the peptide ligand may replace the third loop. For example, a peptide ligand may replace the loop residues corresponding 58 to 64 of SEQ ID NO: 1 and SEQ ID NO: 3.

In some embodiments, a peptide ligand of more than 4 residues may be added to the scaffold sequence without replacing scaffold residues. A peptide ligand of 4 or fewer residues may replace the corresponding number of residues in the CKS scaffold sequence.

In some embodiments, a grafted CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 6 or a variant thereof.

(SEQ NO: 6; where [Xi, X2...X _n ] is a peptide ligand of n amino

acids, where n is 0-30 and Xi, X2...X _n are independently any amino acid, for example, independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine, phospho-serine, phospho-threonine and phospho- tyrosine, acetylated amino acids.

In some embodiments, the Skp2-binding region of the grafted CKS scaffold may be replaced by a helical peptide ligand. This may alter the binding specificity of the grafted CKS scaffold. For example, the helical peptide ligand may bind to a target molecule other than Skp2. For example, a grafted CKS scaffold may comprise a peptide ligand in the helix portion of the CKS scaffold. A helical peptide ligand may be inserted in a helical portion of the CKS scaffold at the position corresponding to residues 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3. for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). A“helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold. In some embodiments, a helical peptide ligand replaces the helix of the CKS scaffold. For example, a peptide ligand may replace residues 40 to 45 of SEQ ID NO: 1 or 3. For example, a peptide ligand may replace one or more of residues S41 , E42 and N45 of SEQ ID NO: 1.

In some embodiments, a grafted CKS scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 40, 41 or 44 of SEQ ID NO: 1 or 3. In some embodiments, a grafted CKS scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 42, 43 or 46 of SEQ ID NO: 1 or 3).

Suitable peptide ligands include helical peptide ligands as described herein.

In some embodiments, a grafted CKS scaffold is created by isomorphic replacement of those residues (for example in SEQ ID NO: 1 one or more of residues S41 , E42 and N45) of the helix portion of the CKS scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand. Preferably, the residues corresponding to E40, W43, R44 of SEQ ID NO: 1 are not replaced.

For example, a helix peptide ligand may comprise the amino acid sequence of SEQ ID NO:

7, a fragment of SEQ ID NO: 7 or a variant of either of these. SEQ ID NO: 7, where Xi to X3 are independently any amino acid any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.)

A grafted CKS scaffold may comprise a CKS scaffold comprising a first peptide ligand inserted within the first loop located at a position corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3 or in the second loop between residues 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3 and a second peptide ligand located within the helical region located at positions corresponding to residues 40 to 45 of SEQ ID NO: 1 or 3.

The CKS scaffold of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the CKS scaffolds.

In other embodiments, a CKS scaffold of a chimeric protein may also display binding activity i.e. the CKS scaffold may mediate binding of the chimeric protein to a first target molecule, such as Skp2. The peptide ligand may mediate binding of the chimeric protein to a second target molecule.

Examples of grafted CKS scaffolds according to the first aspect of the invention are shown in Figure 5C and Table 4.

For example, a grafted CKS scaffold may comprise a CKS scaffold with the amino acid sequence of residues 1 to 73 of SEQ ID NO: 665 (PPX69 of Table 4 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of first loop (loop 1 ; residues 25 to 39 of SEQ ID NO: 665) or third loop (loop 3; residues 58 to 64 of SEQ ID NO: 665) or the helical region (helix 1 ; residues 40 to 45 of SEQ ID NO: 665) of the CKS scaffold. Preferably, the target-binding peptide ligand is located in the first or the third loop. Preferably, the E3 ligase-binding peptide ligand is located in helix 1. Most preferably, the E3 ligase-binding peptide ligand is the endogenous SCF ^Skp2 binding sequence of CKS1 which is present in the helical region (helix 1). For example, the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the helix or the target-binding peptide ligand may be in the third loop and the E3 ligase-binding peptide ligand may be in the helix. Suitable target-binding peptide ligands include bcatenin binding ligands, for example peptides from ARC (Adenomatous polyposis coli), such as SEELEALEALELDE and variants thereof, and peptides from SOX, such as DDIEFDQYL and variants thereof, and KRAS binding ligands, for example peptides from KBL, such as PLYISY and variants thereof, peptides from alpha farnesyl transferase, such as ENPKQYN and variants thereof, peptides from beta farnesyl transferase, such as DAYECLDASRPW or KSRDFYH and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS and variants thereof.

In some embodiments, a grafted CKS scaffold of the first aspect may comprise an amino acid sequence shown in Table 4 (SEQ ID NOs: 660-682) or a variant of an amino acid sequence shown in Table 4. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 4 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the CKS scaffold sequence in a reference amino acid sequence of Table 4 is replaced by a different CKS scaffold sequence. Suitable CKS scaffold sequences are described above.

(ii) Coiled-coil Scaffolds

A second aspect relates to chimeric proteins that comprise coiled-coil scaffolds (i.e. grafted coiled-coil scaffolds). One or more peptide ligands are located in the coiled-coil scaffold of the chimeric protein, for example at positions between residues 55 to 57 or between residues 17 to 54 or between residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13 or 14.

Coiled-coil domains are well-known and well-characterized example in the prior art. Coiled- coil domains are among the most extensively used model systems in the area of protein folding and design. A coiled-coil domain is a structural fold in proteins in which 2-7 alpha- helices are coiled together like the strands of a rope. Many coiled-coil-type proteins are involved in important biological functions such as the regulation of gene expression, e.g. transcription factors (Chembiochem. 2004 Feb 6;5(2):170-6 PNAS October 17, 2006 103 (42) 15457-15462; Science. 262 (5138): 1401-7). The invention adopts the well understood sequence/structure relationships of coiled-coil domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a“coiled-coil scaffold” is a peptidyl structure composed of at least two alpha- helices coiled together like the strands of a rope. A coiled-coil may be a dimer or a trimer (of alpha-helices), or may include or consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or up to 12 helices.

The alpha-helices may be parallel or anti-parallel, and may adopt a left or right handed supercoil. Left-handed supercoils maybe preferred in some embodiments.

A coiled-coil scaffold contains a heptad repeat, which is a repeated pattern of hydrophobic (H) or charged (C) amino acid residues, and the pattern may include the heptad HxxHCxC (to specify each position, the heptad is labelled abcdefg, where“a” and“d” are hydrophobic positions). It may be in some embodiments that“a” and“d” are Isoleucine, leucine or valine. A coiled-coil scaffold, as used herein, has a length in the amino acid range of 1-50, 1-20, 1- 1385, and any amino acid length there between. A coiled-coil scaffold is typically 7400 Da in MW, and such scaffolds are in the MW range of 2500 to 158275 Da.

A left-handed coiled-coil scaffold comprises

(i) a heptad repeat, in which the a and d positions are hydrophobic (e.g. leucine, valine, or isoleucine), thus stabilizing helix dimerization through hydrophobic and van der Waals interactions;

(ii) residues e and g are charged (e.g. glutamate or lysine) in order to form inter helical electrostatic interactions. Such interaction patterns should be of the opposite charge in heterodimers to stabilize their interaction, and of the same charge in homodimers to destabilize them; and

(iii) the remaining three positions (b, c, and f) are hydrophilic, as these will form helical surfaces that are exposed to the solvent (Bioessays 2016 Sep; 38(9): 903-916;

Nature. 371 (6492): 80-3).

The length of a coiled-coil scaffold is from 14 amino acids (2 heptad repeats) to 140 amino acids (20 heptad repeats) in length, or may be 21 - 105 amino acids, 28 - 100 amino acids, 35 - 70 amino acids, and any lengths in between the stated length ranges. A coiled-coil scaffold serves as a molecular spacer with respect to the grafted peptide ligands. With respect to the physical size of a coiled-coil, the size is in the range of 40 - 90nm in length, and may be 50nm - 75nm.

A coiled-coil scaffold may comprise helices with repeating sequences which exhibit distinct amphipathic character, with both hydrophobic and polar faces. A coiled-coil scaffold may comprise two (or more) helices which associate via their hydrophobic faces, which then drives coiled-coil formation.

In a water-filled environment (e.g., a cell cytoplasm or a body fluid, or a pharmaceutical composition), thermodynamically, the helices of a coiled-coil scaffold may arrange such that the hydrophobic strands wrap against each other and are sandwiched between the hydrophilic amino acid residues of the strands, with tight packing and van der Waals contacts between the side-chains of the“a” and“d” residues leading to higher stability of the coiled-coil scaffold.

A representative coiled-coil scaffold, as used herein, may be 14-140 amino acids in length. Figure 7 depicts a representative coiled-coil scaffold. [PDB code 1CXZ]

A coiled-coil scaffold may be homotypic (same coiled-coils interact) or heterotypic (different coiled-coils interact). Thus, a coiled-coil scaffold may be a homo- or hetero-oligomer, and may be formed from separate chains, or from consecutive helices of the same chain.

A GCN4 coiled-coil is an example of a coiled-coil scaffold. A GCN4 coiled-coil is a 31- amino-acid (which equates to just over four heptads) parallel, dimeric (i.e. , consisting of two alpha-helices) coiled-coil and has a repeated isoleucine (I) and leucine (L) at the“a” and“d” positions, respectively, and forms a dimeric coiled-coil. A trimeric (three alpha-helices) coiled-coil is formed where“a” and“d” are Leucine and Isoleucine, respectively. A tetrameric (four alpha-helices) coiled-coil is formed where both“a” and“d” are Leucine.

In the case of dimeric coiled-coil scaffolds, the presence of a polar residue (in particular asparagine, N) at opposing“a” positions is preferred to facilitate parallel assembly of the coiled-coil. While asparagine is a preferred residue in the opposition“a” positions of a coiled-coil, any polar residue which permits self-complementary hydrogen bonding between these residues is contemplated to be within a coiled-coil scaffold.

Coiled-coils have underlying sequence repeats that govern their assembly, hence coiled- coils can be reliably predicted from primary sequence (J Struct Biol 155: 140-5). An extensive database of genomic and structural information for coiled-coils (J Mol Biol 403: 480-93), and in the periodic table of coiled-coil structures (J Mol Biol 385: 726-32). and the CCp database (Nucleic Acids Res 37: D315-22) of coiled-coil structures are available in art. (http://coiledcoils.chm.bris.ac.uk/ccplus/search/dynamic interface) ( Bioessays 2016 Sep; 38(9): 903-916).

Suitable coiled-coil scaffolds may include the coiled-coil domain selected from the following, without limitation: effector domain of the protein kinase Serine/ threonine- protein kinase N1 (HR1 family, UniProtKB - Q16512 (PKN1 JHUMAN) ), General control protein GCN4 (UniProtKB - P03069 (GCN4_YEAST)), Golgin subfamily B member 1 (UniProtKB - Q14789 (GOGB1_HUMAN), Spindle assembly abnormal protein 6 homolog (UniProtKB - Q6UVJ0 (SAS6_HUMAN)), Nuclear mitotic apparatus protein 1 (UniProtKB - Q 14980

(NUMA1JHUMAN)), Kinetochore protein NDC80 homolog (UniProtKB - 014777

(NDC80_HUMAN)), Structural maintenance of chromosomes protein 1A (UniProtKB - Q 14683 (SMC1AJHUMAN)), DNA repair protein RAD50 (UniProtKB - Q92878

(RAD50 JHUMAN)), Kinesin-1 (UniProtKB - P33176 (KINHJHUMAN)), Myosin-2 (UniProtKB - Q9UKX2 (MYH2JHUMAN)) , Rho-associated protein kinase 1 (UniProtKB - Q 13464

(ROCK1JHUMAN)), Serine/threonine-protein kinase MRCK alpha (UniProtKB - Q5VT25 (MRCKA_HUMAN)), and Serine/threonine-protein kinase MRCK gamma (niProtKB - Q6DT37 (MRCKG_HUMAN)). Other suitable coiled-coil domains are shown in Tables 9-11.

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable coiled-coil scaffolds and it may include without any limitation: 1CXZ, 2ZTA, 1WT6, 2HY6, 2V71 , 2XU6, 4LTB, 4DZM, 5D3A, 5JXC, 5LXN, 5LXO, 5M48, 1 L8D and 3QH9.

Representative coiled-coil Scaffold Sequences

Most of the work done in coiled-coil domains on small dimeric coiled-coil proteins since they are well characterised and are easier to express. Generally these involve grafting interacting residues on to the b, c, and f positions of helices. The present invention contemplates the use of dimeric and multimeric coiled-coil domains to generate coiled-coil scaffolds that comprise one or more peptide ligands. The coiled-coil scaffolds may comprise amino terminal peptide ligands (N- terminal degrons) and/or carboxy terminal peptide ligands (C- terminal degrons.

GCN4 homodimeric parallel coiled-coil (CC) (PDB ID 2ZTA)

The MDM2 interacting residues of p53TAD have been grafted on to a coiled-coil scaffold to create p53LZ2. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro

(PMID:24804811).

Designed dimeric parallel coiled-coils.

The invention contemplates a homo-dimeric parallel coiled-coil scaffold (for example as in CC-Di (4dzm)) and derivatives of it that can hetero-dimerise. The NOXA-B sequence was grafted on to the designed homodimeric parallel coiled-coil CC-Di (4dzm) to produce an inhibitor of the MCL-1/BID complex (PMID: 30393526). The invention also contemplates generation of hetero-dimer coiled-coil scaffolds (for example CC-Di-A/CC-Di-B) that may have only one copy of the binding motif.

A coiled-coil scaffold may comprise the amino acid sequence of SEQ ID NO: 8 or a variant thereof.

The above coiled-coil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 9 or a variant thereof. The heptad repeat is conserved in coiled-coil domains and is represented as follows

y , p , ubiquitination. For example, the lysine residues in a coiled-coil domain may be replaced by E, N, Q, L, T, S or R to generate a coiled-coil scaffold. A suitable lysine-free coiled-coil scaffold may comprise the amino acid sequence of SEQ ID NO: 8 or a variant thereof.

Table 7 illustrates a representative example of coiled-coil domains in various proteins with details on sequence conservation, length conservation (Leonard et al Bioessays 2016 Sep; 38(9): 903-916)

In some embodiments, coiled-coil-scaffold may have 2-3 lysine residues. In some

embodiments, all lysine residues of the coiled-coil scaffold are replaced with non-lysine residues. In some embodiments, residue Lys36 of a coiled-coil scaffold is replaced with Arginine. In some embodiments, residue Lys39 of a coiled-coil scaffold is replaced with Arginine. In some embodiments, residue Lys41 of a coiled-coil scaffold is replaced with Arginine. In the above embodiments, any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold. A lysine-attenuated coiled-coil scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids, may comprise the amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In some embodiments, a lysine-free coiled-coil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 11 or a variant thereof.

In some embodiments, a peptide loop ligand has been inserted into the loop that connects two helices of a coiled-coil scaffold. The peptide ligand is represented by residue X, where n is 0-30 and X is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,

hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline,

pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.

In some embodiments, a peptide loop ligand has been inserted into the loop that connects two helices of a lysine-free coiled-coil scaffold. The peptide ligand is represented by residue X, where n is 0-30 and X is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline,

pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.

In some embodiments, a coiled-coil scaffold comprising a peptide ligand may be encoded by a nucleic acid sequence of SEQ ID NO: 14 or a variant thereof.

where n is 0-30 and Y is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 5. For instance, Y is selected from the group of codons“att, ate, and ata” if X of SEQ ID NO: 5 were to be Isoleucine. Similarly Y is selected from the group of codons“ett, etc, eta, ctg, tta, ttg” if X of SEQ ID NO: 5 were to be Leucine.

In some embodiments, a lysine-free coiled-coil scaffold comprising a peptide ligand may be encoded by a nucleic acid sequence of SEQ ID NO:8 or a variant thereof.

where n is 0-30 and Y is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 6. For instance, Y is selected from the group of codons“cgt, ege, ega, egg, aga, agg” if X of SEQ ID NO: 12 were to be Arginine. Similarly Y is selected from the group of codons“aat, aac” if X of SEQ ID NO: 13 were to be Asparagine. Table 8 provides codons for each amino acid that can be used to construct a suitable nucleic acid sequence that encodes variants of any of aforesaid nucleic acid sequences. The a-1 helix of a coiled-coil scaffold is located within the positions corresponding to residues 16 (the CC heptad repeat starts at 26) to 54 of SEQ ID NO: 8 or residues 16 to 54 of SEQ ID NO: 10 or residues 16 to 54 of SEQ ID NO: 12, or residues 16 to 54 of SEQ ID NO: 13.

The a-2 helix of a coiled-coil scaffold is located within the positions corresponding to residues 59 to 82 of SEQ ID NO: 8 or 59 to 83 of SEQ ID NO: 10, or residues 59 to 83 of SEQ ID NO: 12 or residues 59 to 83 of SEQ ID NO: 13.

The disordered regions or the loop regions of a coiled-coil scaffold that connect the alpha helices are located within the positions corresponding to residues 55 to 58 of SEQ ID NO: 8 or residues 55 to 58 of SEQ ID NO: 10 or residues 55 to 58 of SEQ ID NO: 12, or residues 55 to 58 of SEQ ID NO: 13.

Grafted coiled-coil Scaffold

In some embodiments, a grafted coiled-coil scaffold comprises a peptide ligand within a loop (a“loop peptide”). For example, a peptide ligand may be inserted between residues 10 to 12 of SEQ ID NO: 8, 10, 12 or 13 or residues 11 to 13 of SEQ ID NO: 702 (loop 1) residues 55 to 58 of SEQ ID NO: 8, 10, 12 or 13 or residues 55 to 57 of SEQ ID NO: 702 (loop 2).

In some embodiments, loop 1 may be located in the N terminal tail of the coiled-coil scaffold.

In some embodiments, a grafted coiled-coil scaffold comprises a peptide ligand within a helix (a“helix peptide”) (for example, inserted between residues corresponding to residues 16 to 54 or residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13).

For example, a peptide ligand may be inserted between residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702 (helix 1) or residues 62 to 69 of SEQ ID NO: 8, 10, 12 or 13 or residues 62 to 70 of SEQ ID NO: 702 (helix 2).

In some embodiments, a grafted coiled-coil scaffold comprises a first peptide ligand between positions 16 and 54 of the coiled-coil scaffold of SEQ ID NO: 8 or 10 or 12 or 13 or between residues 25 to 36 of the coiled-coil scaffold of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of the coiled-coil scaffold of SEQ ID NO: 702. In some embodiments, a grafted coiled-coil scaffold comprises a second peptide ligand inserted between the positions 55 and 58 of the coiled-coil scaffold of SEQ ID NO: 8 or 10 or 12 or 13 or residues 55 to 57 of the coiled-coil scaffold of SEQ ID NO: 702 (loop 2).

In some embodiments, a grafted coiled-coil scaffold comprises a first peptide ligand and a second peptide ligand in relative positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted coiled-coil scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e., a target protein and an E3 ligase.

In some embodiments, a grafted coiled-coil scaffold comprises a first peptide ligand within a first or second helix described herein (for example, inserted or replaced at position between residues 16 to 54 of SEQ ID NO: 8 or 10 or 12 or 13, or residues 23-37 or 62 to 70 of SEQ ID NO: 72) and a second peptide ligand within a first or second loop (for example, inserted or replaced at position between residues 55 and 58 of SEQ ID NO: 8 or 10 or 12 or 13 or residues 11 to 13 or 55 to 57 of SEQ ID NO: 702).

In some embodiments, a grafted coiled-coil scaffold comprises a first helical peptide ligand within a first helix (helix 1); for example, inserted or replaced at position between residues 16 to 54 of SEQ ID NO: 1 , 3 or 5-7 or residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702 and a second helical peptide ligand within a second helix (helix 2); for example, inserted or replaced at position between residues 66 to 77 or residues 62 to 69 of SEQ I D NO: 8 or 10 or 12 or 13 or residues 62 to 70 of SEQ ID NO:

702.

In some embodiments, a grafted coiled-coil scaffold is created by grafting a first helical peptide ligand onto a first helix at a position defined for instance by residues 16 to 54 of SEQ ID NO: 8 or 10 or 12 or 13; or residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702.

In some embodiments, a grafted coiled-coil scaffold is created by grafting a second helical peptide ligand onto a second helix at a position defined for instance by residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13; residues 62 to 69 of SEQ ID NO: 8 or 10 or 12 or 13 or residues 62 to 70 of SEQ ID NO: 702.

In some embodiments, a grafted coiled-coil scaffold is created by isomorphic replacement of solvent exposed residues of a loop (for example, inserted or replaced at position between residues 55 to 58 of SEQ I D NO: 8 or 10 or 12 or 13, or residues 11 to 13 or 55 to 57 of SEQ ID NO: 702) with solvent facing residues of the loop peptide ligand.

In some embodiments, a grafted coiled-coil scaffold is created by isomorphic replacement of those residues (for example, at position between residues 55 to 57 of SEQ ID NO: 8 or 10 or 12 or 13) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand. A grafted coiled-coil scaffold comprises a loop peptide ligand in a loop portion of the coiled- coil scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a coiled-coil domain generally show a high occurrence of insertions across the HMM logo. The loops connecting helices sheets are ideal places for loop grafting of peptide ligands because the loop regions rarely have any conserved residues.

A grafted coiled-coil scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the coiled-coil scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to position at residue 54, 55, 56 or 57 of SEQ ID NO: 8 or 10 or 12 or 13, or residues 11 , 12 or 13 or residues 55, 56 or 57 of SEQ ID NO: 702

A loop peptide ligand may be inserted into the coiled-coil scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to position at residue 56, 57, 58 and 59 of variants of SEQ ID NO: 8 or 10 or 12 or 13 or residues 11 , 12 or 13 or residues 55, 56 or 57 of SEQ ID NO: 702.

A grafted coiled-coil scaffold comprises a helical peptide ligand in a helical portion of the coiled-coil scaffold, for example, isomorphically replaced into a specific location (for example, at position between residues 16 to 54 or at position between residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13 or at position between residues 23 to 37 or at position between residues 62 to 70 of SEQ ID NO: 702) of a helix (thus preserving the existing helical structure). The long helix in a coiled-coil scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.

A grafted coiled-coil scaffold also may contain a peptide ligand within a helical region of the scaffold (i.e., a“helical peptide” is a peptide which is positioned in a helical structure of the scaffold.

In some embodiments, a grafted coiled-coil scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to position at residue 83 of variants of SEQ ID NO: 8 or 10 or 12 or 13.

In some embodiments, a grafted coiled-coil scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 16 of variants of SEQ ID NO: 8 or 10 or 12 or 13. Figure 9 shows an example of coiled-coil scaffold created by a grafting two helical peptide ligands in two helix regions. The first peptide ligand binds to beta-catenin and the second peptide ligand binds to MDM2-amino terminal domain. Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera.

Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between beta- catenin, the coiled-coil scaffold and the Mdm2 E3 ligase is predicted by the structural alignment between the modelled helix grafting and the crystal structure of the p53 degron peptide (sequence FAAYWNLLSAYG) bound to the N-terminal domain of Mdm2.

For example, a coiled-coil scaffold may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands. For example, aromatic residues in the coiled-coil scaffold may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of coiled-coil scaffold sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.

In some embodiments, lysine residues in the coiled-coil scaffold may be replaced by a different residue, such as arginine to prevent unwanted ubiquitination and subsequent degradation. This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC).

The coiled-coil scaffolds of a chimeric protein of the second aspect may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the coiled-coil scaffolds.

Examples of grafted coiled-coil scaffolds according to the second aspect of the invention are shown in Figure 10C and Tables 5 and 12.

For example, a grafted coiled-coil scaffold may comprise a coiled-coil scaffold with the amino acid sequence of residues 1 to 94 of SEQ ID NO: 702 (PPX59 of Table 12 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of first helix (helix 1 ; residues 23 to 37 of SEQ ID NO: 702), second helix (helix 2; residues 62 to 70 of SEQ ID NO: 702), first loop (loop 1 ; residues 11 to 13 of SEQ ID NO: 702) and second loop (loop 2; residues 55 to 57 of SEQ ID NO: 702) of the coiled- coil scaffold. Preferably, the target-binding peptide ligand is located in the first helix or the first loop. Preferably, the E3 ligase-binding peptide ligand is located in the second loop or second helix. For example, the target-binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second helix or the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop. Suitable target-binding peptide ligands include b-catenin binding ligands, for example helical beta-catenin binding sequence from the protein AXIN, such as ILxxHV and variants thereof, and peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE or GSEELEALEALELDEA and variants thereof, and KRAS binding ligands, for example alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof; Suitable E3 ligase-binding peptide ligands include SCF ^Skp2 binding sequences from p27, such as

AGSNEQEPNR and variants thereof, Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL and variants thereof, E3 Cul3-SPOP binding sequences from Puc, such as DEVTSTTSS, and variants thereof, SIAH binding sequences from PHYL, such as

LRPVAMVRPWVR, and variants thereof, COP1 binding sequences from Trib, such as SDQIVPEYQE, and variants thereof, UBR5 binding sequences from PAM2, such as

LSVNAPEFYP, and variants thereof, beta-TRCP binding sequences from CDC25B, such as TEEDDGFVDI, and variants thereof, and MDM2 binding sequences from p53, such as FSxxWxxL and variants thereof.

In some embodiments, a grafted coiled-coil scaffold of the second aspect may comprise an amino acid sequence shown in Table 12 (SEQ ID NOs: 683-706) or a variant of an amino acid sequence shown in Table 12. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 12 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the coiled-coil scaffold sequence in a reference amino acid sequence of Table 12 is replaced by a different coiled-coil scaffold sequence. Suitable coiled-coil scaffold sequences are described above.

(iii) Affibody Scaffolds

A third aspect relates to chimeric proteins that comprise Affibody scaffolds (i.e. grafted Affibody scaffolds). One or more peptide ligands are located in the Affibody scaffold of the chimeric protein, for example at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID NO: 16, 18, and 20 to 53. Affibody is a well-known and well-characterized example of a structural fold. Affibodies are among the most extensively used model systems in the area of protein folding and design. Affibody molecules were originally derived from the B-domain in the immunoglobulin-binding region of staphylococcal protein A. The B-domain is a relatively short cysteine- free peptide of 58 amino acids that is folded into a three-helical bundle structure. The engineered Z- domain retained its affinity for the Fc part of the antibody while the weaker affinity for the Fab region was almost completely lost. (FEBS Letters 584 (2010) 2670-2680).

The invention adopts the well understood sequence/structure relationships of Affibody domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, an“Affibody scaffold” refers to a polypeptide which is composed of one or more three-helix bundles. An Affibody scaffold with Z domain may be preferred over an Affibody scaffold with B domain.

An Affibody scaffold, as used herein, is 30-90 amino acids in length, such as 35-85, 40-80, 50-70, and 55-65 amino acids in length, e.g., 58 amino acids in length. An Affibody scaffold has a molecular weight in the range of 4 kDa - 10 kDa, 5 - 9 kDa, 6 - 8 kDa, or about 6.7 kDa.

A representative Affibody scaffold, as used herein, may be 58 amino acids in length. Fig. 12 depicts a representative Affibody scaffold. (PDB code -2KZJ). Suitable Affibody scaffolds may include the Affibody domain selected from the sequences (SEQ ID NO: 20 to 53) in Tables 14 and 15 without limitation:

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable Affibody scaffolds, and it may include without any limitation: 2KZI, 2KZJ, 3MZW, 2M5A, 1 H0T, 1 LP1 , 5EFW, 2B87, 2B89 and 20TK, 5U3D 5U5F 5U5M 5U6A 1ZXG 2B88 1 DEE 1 DEE 5U4Y 5U4Y 4HJG 4HKZ 4IOI 5CBO 5H7C 5H79 5H7A 5H7B 4NPF 5H7C 5H7A 5H7A 5H7A 5H7B 5H7B 5H79 1 BDC 1 BDD 1Q2N 1SS1 2JWD 2SPZ 4NPF 5H7C 1 FC2 3MZW 5CBN 5COC 5XBY 5EWX 5H7B 5H77 5H77 5H75 5H76 5H77 5H7D 5X3F 4NPD 4NPE 4VWVI 5H7C 4WWI 4ZMD 4WWI 5COC 4ZNC 5H7A 5H7B 1 EDI 1 EDJ 1 EDK 1 EDL 5H79 5H7C Figure 13 shows the alignment of a representative examples of Affibody scaffold from which a consensus sequence is obtained. Table 15 shows the alignment score with consensus sequence as the query with a representative sequence of the Affibody scaffold as the subject. Similar comparisons may be done with other representative sequences to obtain relative residue positions and alignment scores. Figure 14 shows a representative example of a grafted Affibody scaffold with peptide ligands grafted on to them, indicated by shaded loops. A threading program such as SwissModeller (Nucleic Acids Res. 46 (W1), W296- W303 (2018)) and an ab initio folding program such as l_tasser (Protein structure and function prediction. Nature Methods, 12: 7-8 (2015)) or Robetta (Nucleic Acids Research,

Vol. 32, No. S2. (2004), pp. W526-W531) may be used to create models of grafted scaffolds

Suitable Affibody scaffolds include the Affibody domains shown in Figure 13 and Tables 14- 16 (SEQ ID NOs: 16, 18, 20 to 53 and residues 1 to 66 of SEQ ID NO: 715) or variants thereof.

Suitable positions for peptide ligand insertions or replacements for the Affibody scaffolds for variants of sequence listed in Figure 13 and Tables 14 and 15 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID 16, 18, and 20 to 53.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID Nos: 16, 18, and 20 to 53 of the Affibody scaffold or between residues 5 to 16 (helix 1), 23 to 37 (helix 2), 20 to 22 (loop 1) or 37 to 41 (loop 2) of SEQ ID NO: 715.

Figure 15 shows a model of Affibody scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, Affibody scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl

An Affibody scaffold may comprise the amino acid sequence of SEQ ID NO: 16 or a variant thereof

The above Affibody scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 17 or a variant thereof.

Preferred Affibody scaffolds lack lysine residues, for example, to avoid unwanted

ubiquitination. For example, the lysine residues in an Affibody domain may be replaced by (Glutamic acid) E, (Asparagine) N, (Aspartic acid) D, (Glutamine) Q, (Leucine) L, (Threonine) T, (Serine) S, Valine (V), or (Arginine) R to generate an Affibody scaffold. A suitable lysine- free Affibody scaffold may comprise the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

In some embodiments, an Affibody scaffold may have 4-5 lysine residues. In some embodiments, all lysine residues of an Affibody scaffold are replaced with non-lysine residues. In some embodiments, residue Lys4 of an Affibody scaffold is replaced with Aspartic acid or Asparagine. In some embodiments, residue Lys7 of an Affibody scaffold is replaced with glutamic acid or threonine. In some embodiments, residue Lys27 of an Affibody scaffold is replaced with glutamine, glutamic acid or Arginine. In some

embodiments, residue Lys49 forms a salt bridge with residue Glu15 at a1 helix of an

Affibody scaffold and maybe replaced with Valine or Leucine. In some embodiments, residue Lys50 of an Affibody scaffold is replaced with arginine or glutamine. In some embodiments, residue Lys58 at the C-terminus of an Affibody scaffold is replaced with a residue selected from a group consisting of glutamine, arginine or aspartic acid. In some embodiments, residue Glu15 maybe mutated to Gln15 to enable better hydrophobic packing with residue Val49. In the above embodiments, any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold. A lysine-attenuated Affibody scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids, may comprise the amino acid sequence of SEQ ID NO: 18 or a variant thereof.

NO: 18; positions of replaced K residues bolded and underlined)

In some embodiments, a lysine-attenuated Affibody scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 19 or a variant thereof.

The a-helix 1 (a1) of an Affibody scaffold is located within the positions corresponding to residues 5 to 19 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and 5 to 16 of SEQ ID NO: 715 (helix 1).

The a-helix 2 (a2) of an Affibody scaffold is located within the positions corresponding to residues 23 to 37 of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NOs: 20 to 53 and SEQ ID NO: 715 (helix 2).

The a-helix 3 (a3) of an Affibody scaffold is located within the positions corresponding to residues 40 to 56 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53.

The disordered regions or the loop regions of an Affibody scaffold that connect the a-helices are located within the positions corresponding to residues 20 to 22 and 38 to 39 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53.

The loop 1 of an Affibody scaffold that connects the a-helices a1 and a2 is located within the positions corresponding to residues 20 to 22 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and SEQ ID NO: 715.

The loop 2 of an Affibody scaffold that connect the a-alpha a2 and a3 are located within the positions corresponding to residues 38 to 39 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and residues 37 to 41 of SEQ ID NO: 715.

Grafted Affibody Scaffold

In some embodiments, a grafted Affibody scaffold comprises a peptide ligand within a loop (a“loop peptide”) (for example, inserted into or replaced at a position between residues 20 to 21 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53).

In some embodiments, a grafted Affibody scaffold comprises a peptide ligand within a helix (a“helix peptide”) (for example, inserted into or replaced at a position between residues 40 to 56 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53).

In some embodiments, a grafted Affibody scaffold comprises a first and a second peptide ligands in two different loops, or at the two different termini of the scaffold, or in a loop and at a terminus of the scaffold.

In some embodiments, a grafted Affibody scaffold comprises a peptide ligand in the loop 1 (for example, inserted into or replaced at a position between residues 20 to 22 of SEQ ID NO: 16 or 18 or 20 to 53 or 715) that connect the a1 and a2 helices.

In some embodiments, a grafted Affibody scaffold comprises a peptide ligand in the loop 2 (for example, inserted into or replaced at a position between residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 37 to 41 of SEQ ID NO: 715) that connect the a2 and a3 helices.

In some embodiments, it is preferable for a grafted Affibody scaffold to comprise a peptide ligand in the loop 1 (residues 20 to 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) that connect the a1 and a2 helices rather than the loop 2 (residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 37 to 41 of SEQ ID NO: 715) that connects the a2 and a3 helices, as grafting onto loop 1 may have less impact on the thermodynamic stability of the scaffold.

In some embodiments, a grafted Affibody scaffold comprises a first peptide ligand in the loop 1 (residues 20 and 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) and a second peptide ligand in the loop 2 (residues 38 and 39 of SEQ ID NO: 16 or 18 or 20 to 53 residues 37 to 41 of SEQ ID NO: 715).

In some embodiments, a grafted Affibody scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and relative orientations such that the two peptide ligands do not interfere sterically with each other and in such a way that they are displayed such that they face in opposite directions to each other. This type of relative arrangement ensures that the grafted peptides of the scaffold can each interact with their cognate binding partners, i.e. , a target protein and an E3 ligase.

In some embodiments, a grafted Affibody scaffold comprises a first peptide ligand grafted onto a loop (e.g. residues 20 to 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) and a second peptide ligand grafted onto a helix (e.g. residues 40 to 46 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 5 to 16 or 23 to 37 of SEQ ID NO: 715).

In some embodiments, a grafted Affibody scaffold comprises a first helical peptide ligand grafted onto a first helix (e.g. residues 5 to 19 of SEQ ID NO: 16 or 18 or 20 to 53; residues 5 to 16 of SEQ ID NO: 715) and a second helical peptide ligand grafted onto a second helix (e.g. residues 40 to 56 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 37 to 41 of SEQ ID NO: 715).

In some embodiments, a grafted Affibody scaffold is created by grafting a first helical peptide ligand onto a first helix at a position defined for instance by residues 5 to 19 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 5 to 16 of SEQ ID NO: 715.

In some embodiments, a grafted Affibody scaffold is created by grafting a second helical peptide ligand onto a second helix at a position defined for instance by residues 40 to 56 or 23 to 37 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 23 to 37 of SEQ ID NO: 715.

In some embodiments, it is preferable to replace the solvent-exposed residues of an Affibody scaffold with the outward-facing residues of a peptide ligand.

In some embodiments, a grafted Affibody scaffold is created by isomorphic replacement of solvent exposed residues of a loop (e.g residues 20 to 22 or residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) or in some embodiments a grafted Affibody scaffold is created by insertion of residues within the loop (e.g. between residues 20 to 22 or between residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) with solvent facing residues of the loop peptide ligand.

In some embodiments, a grafted Affibody scaffold is created by isomorphic replacement of those residues (e.g. residues 20 to 22 or residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand.

A grafted Affibody scaffold comprises a loop peptide ligand in a loop portion of the Affibody scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in an Affibody domain generally show a high occurrence of insertions across the HMM logo. The loops connecting the alpha-helices are ideal places for loop grafting of peptide ligands because the loop regions do not have any conserved residues.

A grafted Affibody scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the Affibody scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 20 of SEQ ID NO: 16 or 18 or 20 to 53 or 715. A loop peptide ligand may be inserted into the Affibody scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 21 of variants of SEQ ID NO: 16 or 18 or 20 to 53 or 715.

A grafted Affibody scaffold comprises a helical peptide ligand in a helical portion of the Affibody scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). The long helix in Affibody scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.

A grafted Affibody scaffold also may contain a peptide ligand within a helical region of the scaffold (i.e., a“helical peptide” is a peptide which is positioned in a helical structure of the scaffold. In some embodiments, a grafted Affibody scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 40 of variants of SEQ ID NO: 16 or 18 or 20 to 53.

In some embodiments, a grafted Affibody scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 41 of variants of SEQ ID NO: 16 or 18 or 20 to 53.

In some embodiments, a grafted Affibody scaffold may contain a helical peptide ligand immediately after a proline residue in a helix.

Examples of grafted Affibody scaffolds according to the third aspect of the invention are shown in Figure 16B and Tables 13 and 16.

For example, a grafted Affibody scaffold may comprise an Affibody scaffold with the amino acid sequence of residues 1 to 66 of SEQ ID NO: 715 (PPX86 of Table 16 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first helix (helix 1 ; residues 5 to 16 of SEQ ID NO: 715), second helix (helix 2; residues 23 to 37 of SEQ ID NO: 715), first loop (loop 1 ; residues 20 to 22 of SEQ ID NO: 715), and second loop (loop 2; residues 37 to 41 of SEQ ID NO: 715) of the Affibody scaffold. Preferably, the peptide ligand is located in any of the first or second helix or first loop of the Affibody scaffold. Preferably, the E3 ligase-binding peptide ligand is located in the second helix or second loop of the Affibody scaffold. For example, the target binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the second helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop or the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second helix. Suitable target-binding peptide ligands include b-catenin binding ligands, for example helical beta-catenin binding sequence from the protein AXIN, such as ILxxHV, AxxILDXHV or ILDxHV and variants thereof, peptides from BCL9, such as TLxxlQxxL, LxTLxxlQ, and SLxxlxxML and variants thereof, and peptides from ARC

(Adenomatous polyposis coli), such as SEELEALEALELDE, SEELEALEALELDEAS or GGSEELEALEALELDEASGS and variants thereof, and KRAS binding ligands, for example alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE or IxxTNxxKTXE and variants thereof, and peptides from RBP, such as

SHYPWFKARLYPLS, GHYPWFKARLYPLA, GGSHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof; Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLS and GLDPETGELLG and variants thereof.

In some embodiments, a grafted Affibody scaffold of the third aspect may comprise an amino acid sequence shown in Table 16 (SEQ ID NOs: 707-715) or a variant of an amino acid sequence shown in Table 16. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 16 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the Affibody scaffold sequence in a reference amino acid sequence of Table 16 is replaced by a different Affibody scaffold sequence. Suitable Affibody scaffold sequences are described above.

The Affibody scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Affibody scaffolds.

(iv) Trefoil Scaffolds

A fourth aspect relates to chimeric proteins that comprise Trefoil scaffolds (i.e. grafted Trefoil scaffolds). One or more peptide ligands are located in the Trefoil scaffold of the chimeric protein, for example at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of SEQ ID NOs: 54, 56 or 28 to 78) of SEQ ID NOs: 54, 56 or 28 to 78.

Trefoil domains are among the most extensively used model systems in the area of protein folding and design. (Proc Natl Acad Sci U S A. 2013 Feb 5;110(6) :2135-9). A detailed analysis of the geometry and architecture of the b-trefoil fold was done by Chothia et al. (J Mol Biol. 1992 Jan 20; 223(2) :531 -43; Protein Sci. 2001 Dec; 10(12): 2587-2599.). A Trefoil domain is composed of six anti parallel beta strands closed off at one end by b-hairpin structures and exhibiting a threefold rotational symmetry at the tertiary structure level.

The invention adopts the well understood sequence-structure relationships of Trefoil domains of proteins, and provides a Trefoil domain as a discrete scaffold onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

Trefoil Scaffolds

A“Trefoil scaffold” is a small, soluble, stable protein that exhibits a threefold rotational symmetry. A Trefoil scaffold is composed of six anti-parallel beta-strands closed off at one end by b-hairpin structures (Fig. 18). A Trefoil scaffold has a length in the amino acid range of 47-140, 140-155, 140-233 amino acids. A Trefoil scaffold is typically 15265Da, and is in the MW range of 5120-15265, 15265-18034, 15270-25520 Da.

A representative Trefoil scaffold is one which is about 140 amino acids in length. Figure 18 depicts a representative Trefoil scaffold, (PDB code 2AFG) which has b strands tilted at -56° to the barrel axis, a barrel diameter of ~16A, and a b- barrel shear number (i.e., the stagger of the strands in the barrel) of 12 (J Mol Biol. 1992 Jan 20; 223(2):531-43.).

The Trefoil domain of Human acidic fibroblast growth factor (FGF-1) is a non-limiting example of a Trefoil scaffold which exhibits a characteristic pseudo-threefold axis of symmetry when viewed down the b-barrel axis. The monomeric structural unit of this threefold symmetry consists of a pair of anti parallel b-sheets, referred to as a b hairpin. This architecture is composed of three repeating“trefoil” subdomains, each of 40-50 amino acids in length and composed of a pair of anti parallel b-hairpin structures. Within the structure are a total of 12 b-strands (numbered #1-12) and 11 reverse turns. (Protein Sci. 201221(12): 1911-1920.)

Suitable Trefoil scaffolds may include the Trefoil domain selected from the following, without limitation: fibroblast growth factors (J Biochem. 1991 110(3):360-3; Science 1991

251 (4989) :90-3), interleukin-1 a and b ( Proc Natl Acad Sci U S A 198986(24) :9667-71.), plant cytotoxins (Proteins 1991 10(3):240-50; J Mol Biol 1995 Jui 14; 250(3) :354-67;

Biochem Biophys Res Commun 1999257(2) :418-24) , bacterial toxins (Nat Struct Biol. 1998 Oct; 5(10).898-902; J Biol Chem 2000275( 12):8889-94) , mannose receptor (J Exp Med 2000 191(7):1105-16), an actin binding protein (Nature 1992359(6398) :855-8), amylase (Structure 1998 6(5):649-59), xylanase (FEBS Lett. 1999460(1 ):61 -6), and Kunitz soybean trypsin inhibitors (Biochemistry 1974 13(20):4212-28; J Mol Biol 1991 Jan 5; 217(1 ):153-76; J Mol Biol 1998275(2) :347 -63), as a dimeric element in the structure of the protease inhibitor ecotine, and as a trimeric arrangement in the b-trefoil hyperfamily (J Mol Evol 2000 50(3):214-23; J Mol Biol 2000302(5) :1041 -7).

SEQ ID NO: 54 is a designed modular protein made of three identical repetitions. The modular repeat unit is blasted in Uniprot and presented in Table 17 with the top 20 best matches. Sequence numbers refer to the position of the Trefoil repeat unit in the context of the full-length protein.

The Consensus sequence from the 20 best hits is determined to be

“lads+DGyvRLiaRHSGKALeVqgASTaDGANvvQYsdwGGdNQqWqlvklgdd gtdpgp”. The upper case indicates high conservation; lower case indicates low conservation; + indicates that not all sequences have a residue at that position.

Suitable Trefoil scaffolds include the Trefoil domains shown in Figure 19 and Tables 17, 19 and 20 (SEQ ID NOs: 54, 55 or 28 to 78 and residues 1 to 149 of SEQ ID NO: 722) or variants thereof.

Suitable positions for peptide ligand insertions or replacements for the Trefoil scaffolds for variants of sequence listed in Figure 19 and Tables 17, 19 and 20 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID 54, 56 or 28 to 78 or 722.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of SEQ ID NO: 54, 56 or 28 to 78. In other embodiments, one or more peptide ligands may be inserted or replaced at positions between residues in a trefoil scaffold corresponding to residues 47 to 49 (loop 1) or residues 116 to 118 (loop 2) of SEQ ID NO: 54, 56 or 58 to 78 or residues 49 to 51 (loop 1) or residues 117 to 119 (loop 2) of SEQ ID NO: 722.

Representative Trefoil Scaffolds Sequences

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable Trefoil scaffolds and it may include without any limitation: 1 PZZ, 1Q03, 1Q04, 2AQZ, 3JUT, 3K1X, 3H6Q, 3H6R, 4I4R, 40W4 and 4XKI.

A Trefoil scaffold may comprise the amino acid sequence of SEQ ID NO: 54 or a variant thereof.

The secondary structure of a representative example of a Trefoil scaffold is shown in Figure 19. The sequence in figure 19 corresponds to SEQ ID NO: 54. The beta-strands represented as arrows and italic text, helices as rectangles and bold text. Unstructured regions connect the secondary structure elements. The helices are very short and are part of the long beta- turns and are not present in all trefoils.

The above Trefoil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 55 or a variant thereof.

Preferred Trefoil scaffolds lack lysine residues, for example, to avoid unwanted

ubiquitination. For example, the lysine residues in a Trefoil domain may be replaced by E, N, Q, L, T, S or R to generate a Trefoil scaffold. A suitable lysine-free Trefoil scaffold may comprise the amino acid sequence of SEQ ID NO: 54 or a variant thereof. In some embodiments, each of the three repeating sub-domains of the trefoil scaffold has two lysine residues. In some embodiments, all six lysines of the Trefoil scaffold are replaced with non-lysine residues. In some embodiments, residue Lys 6 of a first repeating subdomain of a Trefoil scaffold is replaced with Threonine or Arginine. In some

embodiments, residue Lys 14 of a first repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine. In some embodiments, residue Lys53 of a second repeating subdomain of a Trefoil scaffold is replaced with Threonine or Arginine. In some

embodiments, residue Lys 61 of a second repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine. In some embodiments, residue Lys 100 of a third repeating domain of a Trefoil scaffold is replaced with Threonine or Arginine. In some embodiments, residue Lys 108 of a third repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine. In the above embodiments, any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold. A lysine- attenuated Trefoil scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids, may comprise the amino acid sequence of SEQ ID NO: 56 or a variant thereof.

In some embodiments, a lysine-free Trefoil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 57 or a variant thereof.

The b-1 strand of a Trefoil scaffold is located within the positions corresponding to residues 4 to 9 of SEQ ID NO: 54 or 4 to 9 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-2 strand of a Trefoil scaffold is located within the positions corresponding to residues 14 to 18 of SEQ ID NO: 54 or 14 to 18 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78 The b-3 strand of a Trefoil scaffold is located within the positions corresponding to residues 29 to 32 of SEQ ID NO: 54 or 29 to 32 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The b-4 strand of a Trefoil scaffold is located within the positions corresponding to residues 42 to 48 of SEQ ID NO: 54 or 42 to 48 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-5 strand of a Trefoil scaffold is located within the positions corresponding to residues 51 to 56 of SEQ ID NO: 54 or 51 to 56 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-6 sheet of a Trefoil scaffold is located within the positions corresponding to residues 62 to 65 of SEQ ID NO: 54 or 62 to 65 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The b-7 strand of a Trefoil scaffold is located within the positions corresponding to residues 76 to 79 of SEQ ID NO: 54 or 76 to 79 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The b-8 strand of a Trefoil scaffold is located within the positions corresponding to residues 89 to 93 of SEQ ID NO: 54 or 89 to 93 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-9 strand of a Trefoil scaffold is located within the positions corresponding to residues 99 to 103 of SEQ ID NO: 54 or 99 to 103 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-10 strand of a Trefoil scaffold is located within the positions corresponding to residues 109 to 1 12 of SEQ ID NO: 54 or 109 to 112 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The b-1 1 strand of a Trefoil scaffold is located within the positions corresponding to residues 123 to 126 of SEQ ID NO: 54 or 123 to 126 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The b-12 strand of a Trefoil scaffold is located within the positions corresponding to residues 136 to 140 of SEQ ID NO: 54 or 136 to 140 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The b-1 1 strand of a Trefoil scaffold is located within the positions corresponding to residues 123 to 126 of SEQ ID NO: 54 or 123 to 126 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78

The disordered regions or the loop regions of a Trefoil scaffold are located within the positions corresponding to residues 10 to 14 of SEQ ID NO: 54 or 10 to 14 of SEQ ID NO:

56 or of SEQ ID NO: 58 to 78, 23 to 28 of SEQ ID NO: 54 or 23 to 28 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 33 to 36 of SEQ ID NO: 54 or 33 to 36 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 57 to 61 of SEQ ID NO: 54 or 57 to 61 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 70 to 75 of SEQ ID NO: 56 or 70 to 75 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 80 to 83 of SEQ ID NO: 54 or 80 to 83 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 94 to 97 of SEQ ID NO: 54 or 94 to 97 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 104 to 108 of SEQ ID NO: 54 or 104 to 108 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 117 to 122 of SEQ ID NO: 54 or 117 to 122 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, and 127 to 130 of SEQ ID NO: 54 or 127 to 130 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The first loop of the Trefoil scaffold is located within the positions corresponding to residues 10 to 14 of SEQ ID NO: 54 or 10 to 14 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The second loop of the Trefoil scaffold is located within the positions corresponding to residues 23 to 28 of SEQ ID NO: 54 or 23 to 28 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The third loop of the Trefoil scaffold is located within the positions corresponding to residues 33 to 36 of SEQ ID NO: 54 or 33 to 36 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The fourth loop of the Trefoil scaffold is located within the positions corresponding to residues 57 to 61 of SEQ ID NO: 54 or 57 to 61 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78 or 49 to 51 of SEQ I D NO: 722.

The fifth loop of the Trefoil scaffold is located within the positions corresponding to residues 70 to 75 of SEQ ID NO: 54 or 70 to 75 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The sixth loop of the Trefoil scaffold is located within the positions corresponding to residues 80 to 83 of SEQ ID NO: 54 or 80 to 83 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The seventh loop of the Trefoil scaffold is located within the positions corresponding to residues 94 to 97 of SEQ ID NO: 54 or 94 to 97 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The eighth loop of the Trefoil scaffold is located within the positions corresponding to residues 104 to 108 of SEQ ID NO: 54 or 104 to 108 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.

The ninth loop of the Trefoil scaffold is located within the positions corresponding to residues 117 to 122 of SEQ ID NO: 54 or 117 to 122 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78 or 117 to 119 of SEQ ID NO: 722.

The tenth loop of the Trefoil scaffold is located within the positions corresponding to residues 127 to 130 of SEQ ID NO: 54 or 127 to 130 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78,

Grafted Trefoil Scaffold

In some embodiments, a grafted Trefoil scaffold comprises a peptide ligand within a loop (a “loop peptide”).

In some embodiments, a grafted Trefoil scaffold comprises a helical peptide ligand within a loop (a“helix peptide”).

In some embodiments, a grafted Trefoil scaffold comprises a peptide ligand within a b strand (a“strand peptide”).

In some embodiments, a grafted Trefoil scaffold comprises a peptide ligand that is less than or equal to 30 amino acids in length.

In some embodiments, a grafted Trefoil scaffold is created by inserting peptide ligands into one or more of loops or strands of the Trefoil scaffold.

In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand and a second peptide ligand, both of which are loop peptide ligands. In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand which is a helix peptide and a second peptide ligand which is a loop peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand which is a strand peptide and a second peptide ligand which is a loop peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand which is a strand peptide and a second peptide ligand which is a helix peptide ligand.

In some embodiments, a grafted Trefoil scaffold comprises more than one loop peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises more than one strand peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises more than one helix peptide ligand.

In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand between positions 21 and 22 of SEQ ID NO: 54 or 56 or 58 to 78 in the Trefoil scaffold. In some embodiments, a grafted Trefoil scaffold comprises a second peptide ligand between the positions 93 and 94 of the Trefoil scaffold.

In other embodiments, a grafted Trefoil scaffold comprises a first peptide ligand between residues corresponding to residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722 in the Trefoil scaffold. In some embodiments, a grafted Trefoil scaffold comprises a second peptide ligand between the residues

corresponding to residues 116 to 118 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 117 to 119 of SEQ ID NO: 722 of the Trefoil scaffold. In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted Trefoil scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e. , a target protein and an E3 ligase.

In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand within a first loop (residues 21 to 22 of SEQ ID NO: 54 or 56 or 58 to 78) and a second peptide ligand within a second loop (residues 93 to 94 of SEQ ID NO: 54 or 56 or 58 to 78).

In some embodiments, a grafted Trefoil scaffold comprises a first loop peptide ligand in the loop region starting at position 21 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold comprises a second loop peptide ligand in the loop region starting at position 98 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold comprises a first peptide ligand within a first loop (e.g. loop 1 ; residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722) and a second peptide ligand within a second loop (e.g. loop 2;

residues 116 to 118 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 117 to 119 of SEQ ID NO: 722).

For example, a grafted Trefoil scaffold may comprises a first loop peptide ligand between residues of the Trefoil scaffold corresponding to residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722 and a second loop peptide ligand between residues of the Trefoil scaffold corresponding to residues 116 to 118 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 117 to 119 of SEQ ID NO: 722.

A grafted Trefoil scaffold may be created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722

A grafted Trefoil scaffold may be created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722.

In some embodiments, a grafted Trefoil scaffold comprises a first loop peptide ligand between positions 13 and 14 of SEQ ID NO: 54 or 56 or 58 to 78 of the Trefoil scaffold and a second loop peptide ligand between the positions 82 and 83 of SEQ ID NO: 54 or 56 or 58 to 78 of the Trefoil scaffold.

In some embodiments, a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 10 to 14 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 80 to 83 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 35 to 36 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 106 to 107 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 59 to 60 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 129 to 130 of SEQ ID NO: 54 or 56 or 58 to 78.

In some embodiments, a grafted Trefoil scaffold is created by isomorphic replacement of solvent-exposed residues of a loop (for example, residues 21 to 28 of SEQ ID NO: 54 or 56 or 58 to 78) with solvent-facing residues of the loop peptide ligand.

In some embodiments, a grafted Trefoil scaffold is created by isomorphic replacement of those residues (for example, residues 94 to 98 of SEQ ID NO: 54, 56 or 58 to 78) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand.

A grafted Trefoil scaffold comprises a loop peptide ligand in a loop portion of the Trefoil scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a Trefoil domain generally show a high occurrence of insertions across the HMM logo. The loops connecting beta sheets are ideal places for loop grafting of peptide ligands because the loop regions do not have any conserved residues. A grafted Trefoil scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the Trefoil scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 21 of SEQ ID NO: 54 or 56 or 58 to 78. A loop peptide ligand may be inserted into the Trefoil scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 94 of SEQ ID NO: 54 or 56 or 58 to 78.

Examples of grafted trefoil scaffolds according to the fourth aspect of the invention are shown in Figure 21C and Tables 18 and 20.

For example, a grafted trefoil scaffold may comprise a trefoil scaffold with the amino acid sequence of residues 1 to 149 of SEQ ID NO: 722 (PPX93 of Table 20 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first, second, third and fourth loops of the trefoil scaffold. Preferably, the peptide ligand is located in the first or second loop or the helical region. Preferably, the E3 ligase-binding peptide ligand is located in the second or first loop. For example, the target binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop or the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop. Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE, SEELEALEALELDEAS and

GSEELEALEALELDEASGS and variants thereof, peptides from AXIN, such as ILxxHV, AxxILDxHV, ILDVHV, or ILDxHV and variants thereof, peptides from BCL9, such as

TLxxlQxxL, LxTLxxlQ, and SLxxlxxML and variants thereof, and KRAS binding ligands, for example peptides from KBL, such as PLYISY, PLYISYDPV and PLYISYPV and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and HYPWFKARLYPL and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, and GLDPETGELL and variants thereof.

In some embodiments, a grafted trefoil scaffold of the fourth aspect may comprise an amino acid sequence shown in Table 20 (SEQ ID NOs: 716-722) or a variant of an amino acid sequence shown in Table 20. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 20 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the trefoil scaffold sequence in a reference amino acid sequence of Table 20 is replaced by a different trefoil scaffold sequence. Suitable trefoil scaffold sequences are described above.

Figure 20 shows an example of Trefoil -scaffold created by a grafting two loop peptide ligands onto two loop regions. Loop insertions were modelled with MODELLER (Current Protocols in Bioinformatics 54, John Wiley & Sons, Inc., 5.6.1-5.6.37, 2016) and the energy of the resulting proteins was minimized by UCSF Chimera (J Comput Chem. 2004

Oct;25(13):1605-12).

The Trefoil scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Trefoil scaffolds (which have no native functions in binding to protein or DNA).

M PDZ Scaffolds

A fifth aspect relates to PDZ scaffolds (i.e. grafted PDZ scaffolds). One or more peptide ligands are located in the PDZ scaffold of the chimeric protein, for example between residues 20 to 24, or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the PDZ scaffold (SEQ ID NOs: 79 to 85, 89 to 189 and 190 to 290) or between residues 28 to 30, or between residues 48 to 51 or between residues 90 to 91 , or between residues 70 to 80 of the PDZ scaffold (residues 1 to 108 of SEQ ID NO: 727).

A PDZ domain is a common structural domain of 70-110 amino-acids found in signalling proteins of bacteria, yeast, plants, viruses and animals. (PDZ is an initialism combining the first letters of the first three proteins discovered to share the domain— post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1).

The invention adopts the well understood sequence/structure relationships of PDZ domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

A PDZ scaffold is a folded protein composed of 4 to 6 b-strands, one short a-helix and one long a-helix. A PDZ scaffold has a length in the range of 70-110 amino acids or any length there between. A PDZ scaffold is globular and has a molecular weight in the range of 7500 Da to 12,300 Da.

A PDZ scaffold may have a natural binding site located between one of the b-strands and the long a-helix, forming a binding pocket constituted by several hydrophobic amino acids, the main chain atoms of which form a nest (protein structural motif) binding the C-terminal carboxyl ate of the protein or peptide ligand.

Suitable PDZ scaffolds may include the PDZ domains selected from the following, without limitation: Disks large homolog 4 (PSD-95 ;UniProtKB - P78352 (DLG4_HUMAN); Tyrosine- protein phosphatase non-receptor type 13 (PTPN13, PTB-BL, UniProtKB - Q12923

(PTN13_HUMAN)) , Glutamate receptor-interacting protein 1 (UniProtKB - Q9Y3R0

(GRIP1JHUMAN)), Neuroblast differentiation-associated (AHNAK; UniProtKB - Q09666 (AHNK_HUMAN)), Membrane-associated guanylate kinase, VWV and PDZ domain- containing protein 2 (UniProtKB - Q86UL8 (MAGI2_HUMAN)), Amyloid beta A4 precursor protein-binding family A member 1 (APBA1 ; UniProtKB - Q02410 (APBA1 JHUMAN)), Amyloid beta A4 precursor protein-binding family A member 2 (APBA2; UniProtKB - Q99767 (APBA2_HUMAN)), Amyloid beta A4 precursor protein-binding family A member 3 (APBA3; UniProtKB - 096018 (APBA3_HUMAN)), Rho GTPase-activating protein 21 (UniProtKB - Q5T5U3 (RHG21JHUMAN), Rho GTPase-activating protein 23 (UniProtKB - Q9P227 (RHG23_HUMAN) ), Rho guanine nucleotide exchange factor 11 (UniProtKB - 015085 (ARHGB_HUMAN)), Rho guanine nucleotide exchange factor 12 (UniProtKB - Q9NZN5 (ARHGC_HUMAN)), Caspase recruitment domain-containing protein 11 (UniProtKB - Q9BXL7 (CAR11_HUMAN)), Caspase recruitment domain-containing protein 14 (UniProtKB - Q9BXL6 (CAR14_HUMAN)), Peripheral plasma membrane protein (UniProtKB - 014936 (CSKP_HUMAN)), PDZ and LIM domain protein 1 (UniProtKB - 000151 (PDLI1 JHUMAN)), Connector enhancer of kinase suppressor of ras 2 (UniProtKB - Q8WXI2

(CNKR2_HUMAN)), Connector enhancer of kinase suppressor of ras 3 (UniProtKB - Q6P9H4 (CNKR3 JHUMAN)), , Whirlin (UniProtKB - Q9P202 (WHRNJHUMAN)), Discs large homolog 1 (UniProtKB - Q12959 (DLG1 JHUMAN)), Discs large homolog 2 (UniProtKB - Q15700 (DLG2 JHUMAN)), Discs large homolog 3 (UniProtKB - Q92796 (DLG3_HUMAN)), Discs large homolog 5 (UniProtKB - Q8TDM6 (DLG5_HUMAN)), Segment polarity protein dishevelled homolog DVL-1 (UniProtKB - 014640 (DVL1_HUMAN)), Segment polarity protein dishevelled homolog DVL-2 (UniProtKB - 014641 (DVL2_HUMAN)), Segment polarity protein dishevelled homolog DVL-3 (UniProtKB - Q92997 (DVL3_HUMAN)), Erbb2 interacting protein, also known as Erbin (UniProtKB - Q96RT 1 (ERBIN_HUMAN)), FERM and PDZ domain-containing protein 1 (UniProtKB - Q5SYB0 (FRPD1 JHUMAN) ), GIPC PDZ domain containing family, member 1 (UniProtKB - 014908 (GIPC1_HUMAN)), Golgi- associated PDZ and coiled-coil motif-containing protein (UniProtKB - Q9HD26

(GOPC_HUMAN)), Pro-interleukin-16 (UniProtKB - Q14005 (IL16JHUMAN)), InaD- like protein (UniProtKB - Q8NI35 (INADL_HUMAN)), Ras-associating and dilute domain- containing protein (UniProtKB - Q96JH8 (RADILJHUMAN)), LIM domain binding 3

(UniProtKB - 075112 (LDB3_HUMAN)), LIM domain kinase 1 (UniProtKB - P53667

(LIMK1_HUMAN)), Lin-7 homolog A (UniProtKB - 014910 (LIN7AJHUMAN)), LIM domain only protein 7 (UniProtKB - Q8WWI1 (LM07_HUMAN)), E3 ubiquitin-protein ligase LNX1 ( UniProtKB - Q8TBB1 (LNX1_HUMAN) ), Ligand of Numb protein X 2 (UniProtKB - Q8N448 (LNX2_HUMAN)), or variants thereof.

Representative PDZ Scaffold Sequences

Table 24 shows the multiple alignment of representative sequences (SEQ ID NO: 89 to 189) of PDZ domains aligned with SEQ ID NO.79 and the multiple alignment of representative sequences of PDZ domains (SEQ ID NO: 190 to 290) aligned with SEQ ID NO. 80 with the top 100 hits from a BLAST® search. Secondary structural elements are represented by arrows for beta-strands and boxes for alpha helices.

Suitable PDZ scaffolds include the PDZ domains shown in Tables 21 , 22 and 24 (SEQ ID NOs: 79-85, 87, 89 to 189 and 190 to 290) or variants thereof. Other suitable PDZ scaffolds include the PDZ domain of residues 1 to 108 of SEQ ID NO: 727 or variants thereof.

Suitable positions for peptide ligand insertions or replacements for the PDZ scaffolds for variants of sequence listed in Tables 21 , 22, 24 and 25 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID NOs: 79-85, 87, 89 to 290 and 727.

In some embodiments, one or more peptide ligands may be inserted or replaced at loops or helices marked by positions residues 20 to 24; or residues 18 to 54; or residues 19 to 22; or residues 13-17; or residues 13 to 23; or residues 20 to 24; or residues 10 to 15; or residues 20 to 24; or residues 51 to 56; or residues 59 to 65; or residues 30 to 42; or residues 23-34; or residues 32 to 34; or residues 30 to 38; or residues 21 to 31 ; or residues 51 to 56; or residues 69 to 72 ; or residues 76 to 81 ; or residues 51 to 54; or residues 42 to 44; or residues 52 to 56; or residues 45 to 49; or residues 46 to 52; or residues 69 to 72; or residues 57 to 62; or residues 50 to 55; or residues 64 to 71 ; or residues 52 to 62; or residues 63 to 67; or residues 73 to 82 ; or residues 82 to 89 ; or residues 78 to 86 ; or residues 71 to 79 ; or residues 72 to 76 ; or residues 75 to 83 or residues 68 to 76 ; or residues 73 to 82 of SEQ ID Nos: 79-85, 87, 89 to 189 and 190 to 290 of the PDZ scaffold.

In some embodiments, one or more peptide ligands may be inserted or replaced at loops at positions correspond to residues 28 to 30, residues 48 to 51 or residues 90 to 91 of SEQ ID NO: 727 (loops 1 , 2, and 3, respectively) or a helix at a position correspond to residues 70 to 80 of SEQ ID NO: 727 (helix 1).

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 79 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 80 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 81 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 82 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 83 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 84 or a variant thereof.

In some embodiments, a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 85 or a variant thereof.

Table 21 shows the consensus sequence from the SMART database of the PDZ domains. The positions at which loops can be inserted and the maximum number of amino acids that are found in natural variants at these points are shown. At positions in the consensus sequence where there is a lysine that is not a highly conserved position, it is preferable to substitute with a polar or small amino acid, thereby preserving thermostability of the molecule.

The two PDZ domains that show the highest thermodynamic stability (around 60 °C) is the 3 ^rd PDZ domain from PSD-95(SEQ ID NO: 79) and the second PDZ domain from protein tyrosine phosphatase non-receptor type 13 (PTP-BL, SEQ ID NO: 80). Extensive folding studies have been performed on these two PDZ domains and this give us key information necessary for successful protein engineering to create multiple interaction sites. An alignment of each one of these PDZ is shown in Table 22.

Multiple alignment of SEQ ID NOs 79 and 80 is shown in Table 24 with the top 100 hits from a BLAST search. Secondary structural elements are represented by arrows for beta-strands and boxes for alpha helices.

Clustal W multiple alignment of SEQ ID NO 79, 80, 81 , 82, 83, 84, 85 and 87 is shown in Table 22. Beta-strands are boxed with a solid line and alpha helices are boxed with a dashed line. The loop regions suitable for insertion or grafting of molecules are labelled.

Helix residues that can be modified are marked with a # symbol. Beta-strand residues that can be modified are marked with a + symbol. Figure 25 shows a model of PDZ1 scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, PDZ scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl .

In some embodiments, a PDZ scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 86 or a variant thereof.

Preferred PDZ scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in a PDZ domain may be replaced by E, Q or R to generate a PDZ scaffold. For example, a suitable lysine-free PDZ scaffold may be formed by substituting the 3 lysines with E, Q or R in SEQ ID NO: 79 or a variant thereof.

In some embodiments, a lysine- free PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 87 or a variant thereof.

In some embodiments, a lysine-free PDZ scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 88 or a variant thereof.

In some embodiments, a PDZ scaffold or a grafted PDZ SLIM scaffold is 3k82. The PDZ domain of 3k82 has only three Lys residues. In some embodiments, the residues Lys46 and Lys71 of the PDZ domain are replaced with glutamine. In some embodiments, the residues Lys46 and Lys71 of the PDZ domain are replaced with glutamic acid. In some

embodiments, the residues Lys46 and Lys71 of the PDZ domain are replaced with aspartic acid. . In some embodiments, the residues Lys46 and Lys71 of the PDZ domain are replaced with arginine.

The b-1 strand of a PDZ scaffold is located within the positions corresponding to residues 13 to 19 of SEQ ID NO: 79 or 9 to 17 of SEQ ID NO: 80 or 8 to 18 of SEQ ID NO: 81 or 2 to 12 of SEQ ID NO: 82 or 8 to 12 of SEQ ID NO: 83 or 13 to 19 of SEQ ID NO: 84 or 1 to 9 of SEQ ID NO: 85 or 13 to 19 of SEQ ID NO: 87.

The b-2 strand of a PDZ scaffold is located within the positions corresponding to residues 25 to 32 of SEQ ID NO: 79 or 66 to 71 of SEQ ID NO: 80 or 23 to 29 of SEQ ID NO: 81 or 18 to 22 of SEQ ID NO: 82 or 24 to 31 of SEQ ID NO: 83 or 25 to 29 of SEQ ID NO: 84 or 16 to 20 of SEQ ID NO: 85 or 25 to 32 of SEQ ID NO: 87.

The b-3 strand of a PDZ scaffold is located within the positions corresponding to residues 35 to 43 of SEQ ID NO: 79 or 73 to 75 of SEQ ID NO: 80 or 43 to 50 of SEQ ID NO: 81 or 35 to 41 of SEQ ID NO: 82 or 35 to 43 of SEQ ID NO: 83 or 39 to 44 of SEQ ID NO: 84 or 32 to 37 of SEQ ID NO: 85 or 35 to 43 of SEQ ID NO: 87.

The b-4 strand of a PDZ scaffold is located within the positions corresponding to residues 57 to 64 of SEQ ID NO: 79 or 92 to 100 of SEQ ID NO: 80 or 63 to 69 of SEQ ID NO: 81 or 56 to 62 of SEQ ID NO: 82 or 57 to 59 of SEQ ID NO: 83 or 63 to 65 of SEQ ID NO: 84 or 53 to 58 of SEQ ID NO: 85 or 57 to 64 of SEQ ID NO: 87.

The b-5 strand of a PDZ scaffold is located within the positions corresponding to residues 66 to 68 of SEQ ID NO: 79 or 71 to 73 of SEQ ID NO: 81 or 64 to 66 of SEQ ID NO: 82 or 61 to 63 of SEQ ID NO: 83 or 86 to 92 of SEQ ID NO: 84 or 60 to 62 of SEQ ID NO: 85 or 66 to 68 of SEQ ID NO: 87.

The b-6 strand of a PDZ scaffold is located within the positions corresponding to residues 85 to 94 of SEQ ID NO: 79 or 90 to 100 of SEQ ID NO: 81 or 83 to 93 of SEQ ID NO: 82 or 86 to 91 of SEQ ID NO: 83 or 81 to 89 of SEQ ID NO: 85 or 85 to 94 of SEQ ID NO: 87.

The long a helix of a PDZ scaffold is located within the positions corresponding to residues 73 to 82 of SEQ ID NO: 79 or 82 to 89 of SEQ ID NO: 80 or 78 to 86 of SEQ ID NO: 81 or 71 to 79 of SEQ ID NO: 82 or 72 to 76 of SEQ ID NO: 83 or 75 to 83 of SEQ ID NO: 84 or 68 to 76 of SEQ ID NO: 85 or 73 to 82 of SEQ ID NO: 87.

The short a helix of a PDZ scaffold is located within the positions corresponding to residues 47 to 50 of SEQ ID NO: 79 or 55 to 58 of SEQ ID NO: 80 or 55 to 56 of SEQ ID NO: 81 or 45 to 49 of SEQ ID NO: 82 or 47 to 51 of SEQ ID NO: 83 or 49 to 51 of SEQ ID NO: 84 or 41 to

45 of SEQ ID NO: 85 or 47 to 50 of SEQ ID NO: 87.

The third a helix of a PDZ scaffold is located within the positions corresponding to residues 95 to 101 of SEQ ID NO: 79 or 95 to 101 of SEQ ID NO: 87.

The disordered regions or the loop regions of a PDZ scaffold are located within the positions corresponding to residues 20 to 24, 51 to 56 and 69 to 72 of SEQ ID NO: 79 or 18 to 54, 59 to 65 and 76 to 81 of SEQ ID NO: 80 or 19 to 22, 30 to 42, 51 to 54, 57 to 62, 74 to 77 and 87 to 89 of SEQ ID NO: 81 or 13 to 17, 23 to 34, 42 to 44, 50 to 55, 67 to 70 and 80 to 82 of SEQ ID NO: 82 or 13 to 23, 32 to 34, 52 to 56, 64 to 71 and 79 to 85 of SEQ ID NO: 83 or 20 to 24, 30 to 38, 45 to 49, 52 to 62 and 66 to 74 of SEQ ID NO: 84 or 10 to 15, 21 to 31 ,

46 to 52, 63 to 67, 77 to 80 and 92 to 96 of SEQ ID NO: 85 or 20 to 24, 51 to 56 and 69 to 72 of SEQ ID NO: 87.

The first loop of the PDZ scaffold is at a position corresponding to residues 20 to 24 of SEQ ID NO: 79; residues 18 to 54 of SEQ ID NO: 80; residues 19 to 22 of SEQ ID NO: 81 ;

residues 13-17 of SEQ ID NO: 82; residues 13 to 23 of SEQ ID NO: 83; residues 20 to 24 of SEQ ID NO: 84; residues 10 to 15 of SEQ ID NO: 85; or residues 20 to 24 of SEQ ID NO:

87.

The second loop of the PDZ scaffold is at a position corresponding to residues 51 to 56 of SEQ ID NO: 79; residues 59 to 65 of SEQ ID NO: 80; residues 30 to 42 of SEQ ID NO: 81 ; residues 23-34 of SEQ ID NO: 82; residues 32 to 34 of SEQ ID NO: 83; residues 30 to 38 of SEQ ID NO: 84; residues 21 to 31 of SEQ ID NO: 85; or residues 51 to 56 of SEQ ID NO:

87.

The third loop of the PDZ scaffold is at a position corresponding to residues 69 to 72 of SEQ ID NO: 79 ; residues 76 to 81 of SEQ ID NO: 80; residues 51 to 54 of SEQ ID NO: 81 ;

residues 42 to 44 of SEQ ID NO: 82; residues 52 to 56 of SEQ ID NO: 83; residues 45 to 49 of SEQ ID NO: 84; residues 46 to 52 of SEQ ID NO: 85; or residues 69 to 72 of SEQ ID NO: 87.

The fourth loop of the PDZ scaffold is at a position corresponding to residues 57 to 62 of SEQ ID NO: 81 ; residues 50 to 55 of SEQ ID NO: 82; residues 64 to 71 of SEQ ID NO: 83; residues 52 to 62 of SEQ ID NO: 84; or residues 63 to 67 of SEQ ID NO: 7.

In some embodiments, a grafted PDZ scaffold comprises a peptide ligand within a loop, (a “loop peptide”) for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 20 to 24, or in SEQ ID NO: 727 residues 28 to 30, 48 to 51 or 90 to 91.

In some embodiments, a grafted PDZ scaffold comprises a peptide ligand within a helix (a “helix peptide”), for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 73 to 82 or in SEQ ID NO: 727 residues 70 to 80.

In some embodiments, a grafted PDZ scaffold comprises a peptide ligand within a beta- strand (a“strand peptide”), for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 12 to 18.

In some embodiments, a grafted PDZ scaffold comprises a first peptide ligand and a second peptide ligand with relative positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted PDZ scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e. , a target protein and an E3 ligase.

In some embodiments, a grafted PDZ scaffold comprises a first peptide ligand between, for example in SEQ ID NO: 727 positions 28 and 30, positions 48 to 51 or positions 90 to 91 of the PDZ scaffold. In some embodiments, a grafted PDZ scaffold comprises a second peptide ligand inserted between, for example in SEQ ID NO: 727 between positions 70 and 80, of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a first peptide ligand between, for example in SEQ ID NO: 79 positions 20 and 25, of the PDZ scaffold. In some embodiments, a grafted PDZ scaffold comprises a second peptide ligand inserted between, for example in SEQ ID NO: 79 between positions 34 and 35, of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a first helical peptide ligand grafted is between positions 73 and 82 of SEQ ID NO: 79-85, 87, 89-189, 190-290 of the PDZ scaffold and a second helical peptide ligand grafted between the positions 95 and 101 of SEQ ID NO: 79-85, 87, 89-189, 190-290 of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a first helical peptide ligand between, for example in SEQ ID NO: 79, between positions 73 and 82 of SEQ ID NO: 79-85, 87, 89-189, 190-290 of the PDZ scaffold and a first loop peptide ligand between the positions 20 and 24 of SEQ ID NO: 79-85, 87, 89-189, or 190-290 of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a first helical peptide ligand between, for example in SEQ ID NO: 3, between positions 78 and 86 of SEQ ID NO: 79-85, 87, 89-189, 190-290 of the PDZ scaffold and peptide ligand inserted or grafted in loop 2 between the positions 30 and 42 of SEQ ID NO: 79-85, 87, 89-189, 190-290 of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a loop peptide ligand in the third loop between, for example in SEQ ID NO 79: 34 and 35 positions of the PDZ scaffold.

In some embodiments, a grafted PDZ scaffold comprises a first helical peptide ligand between, for example in SEQ ID NO. 79, between positions 73 and 82, of the PDZ scaffold and comprises a second peptide ligand within a beta-strand (a“strand peptide”), for example in SEQ ID NO: 79, between residues 12 to 18.

In some embodiments, a grafted PDZ scaffold comprises a first peptide ligand between, for example in SEQ ID NO: 79 positions 20 and 25, of the PDZ scaffold and comprises a second peptide ligand within a beta-strand (a“strand peptide”), for example in SEQ ID NO: 79, between residues 12 to 18.

In some embodiments, a grafted PDZ scaffold comprises first a peptide ligand inserted within a first loop, for example in SEQ ID NO: 79, between residues 20 to 24 and a second peptide ligand inserted within a second loop for example in SEQ ID NO:79, between residues 34 to 35.

In some embodiments, a grafted PDZ scaffold comprises a first peptide ligand within a helix for example in SEQ ID NO: 79 between residues 73 to 82 and a second peptide ligand inserted within a loop for example in SEQ ID NO: 79, either in loop 1 , between residues 20 to 34 or in loop 2 between residues 34 to 35.

In some embodiments, a grafted PDZ scaffold is created by inserting a peptide ligand into the PDZ scaffold. In some embodiments, a grafted PDZ scaffold is created by inserting a loop peptide ligand in to a loop at a position defined for instance by residues for example in SEQ ID NO: 79 either between 21 and 22 residues in loop 1 or between residues 34 to 35 residues in loop 2.

In some embodiments, a grafted PDZ scaffold comprises isomorphic replacement of solvent exposed residues of a loop for example in SEQ ID NO: 81 in between residues 30 to 42 in loop 2 with solvent facing residues of the loop peptide ligand. In some embodiments, a grafted PDZ scaffold is created by isomorphic replacement of those residues, for example in SEQ ID NO: 79 (E74, N75, I78, K81 , Q82), of an alpha helix which are solvent-exposed and not buried in the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.

In some embodiments, a grafted PDZ scaffold is created by isomorphic replacement of solvent-exposed beta-strand residues in strand one to create a binding site, for example in SEQ ID NO: 81 , (K18, E20, R22, R24, E26).

In some embodiments, a grafted PDZ scaffold comprises peptide ligand which are at least 12 amino acids in length. In some embodiments, a grafted PDZ scaffold comprises peptide ligands which are less than 12 amino acids in length.

In some embodiments, a grafted PDZ scaffold comprises a helical peptide ligand and a loop peptide ligand. In some embodiments, a grafted PDZ scaffold comprises a helical peptide ;ligand and a strand peptide ligand. In some embodiments, a grafted PDZ scaffold comprises a loop peptide and a strand peptide ligand.

In some embodiments, a grafted PDZ scaffold comprises more than one helical peptide ligand. In some embodiments, a grafted PDZ scaffold comprises more than one loop peptide ligand. In some embodiments, a grafted PDZ scaffold comprises more than one strand peptide ligand.

A grafted PDZ scaffold comprises a loop peptide ligand in a loop portion of the PDZ scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a PDZ domain generally show a high occurrence of insertions across the HMM logo. The loops connecting beta sheets are ideal places for loop grafting of peptide ligands because the loop regions don’t have any conserved residues.

A grafted PDZ scaffold comprises a helical peptide ligand in a helical portion of the PDZ scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). The long helix in a PDZ scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.

In some embodiments, a grafted PDZ scaffold, some residues of the grafted PDZ scaffold are not replaced, for example in SEQ ID NO 79, residues H73, A76, A77, A79, L80 would not be changed as they are not solvent exposed, whereas residues E74, N75, I78, K81 , Q82 are exposed and may be modified to for grafting a peptide ligand.

A grafted PDZ scaffold comprises a peptide ligand in a loop portion of the PDZ scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a PDZ domain generally show a high occurrence of insertions across the HMM logo.

Figure 25 shows an example of bifunctional PDZ created by a grafting a first peptide ligand which is a loop and grafting a second peptide ligand that is a helix. The Figure shows two different views of the modelled structure of the complex of loop-helix-grafted PDZ domain in complex with KRAS and the E2-E3 Keapl complex (subunits shaded). A threading program such as SwissModeller (Nucleic Acids Res. 46(W1), W296-W303 (2018)) and an ab initio folding program such as l_tasser (Protein structure and function prediction. Nature Methods, 12: 7-8 (2015)) or Robetta (Nucleic Acids Research, Vol. 32, No. S2. (2004), pp. W526- W531) may be used to create models of grafted scaffolds that are bound to biological molecules through the grafted peptide ligands.

A grafted PDZ scaffold also may contain a loop peptide ligand which when inserted into the disordered or loop region had become a part of the loop region. For example, a loop peptide ligand may be located in the PDZ scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to a residue of the first, second, third or fourth loop of SEQ ID NO: 79 - 83 or 85 as set out above. A peptide ligand may be inserted into the PDZ scaffold immediately before (i.e.

adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue of the first, second, third or fourth loop of SEQ ID NO: 79 - 83 or 85 as set out above.

A grafted PDZ scaffold also may contain a helical peptide ligand which when inserted into the helix had become a part of the helical structure. In some embodiments, a grafted PDZ scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue of a helix of SEQ ID NO: 79 - 83 or 85 as set out above. In some embodiments, a grafted PDZ scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 89 of SEQ ID NO: 79 - 83 or 85 as set out above.)

The PDZ scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the PDZ scaffolds.

In other embodiments, a PDZ scaffold itself may display binding activity, independent of grafting to a chimeric protein. Such a PDZ scaffold, when grafted, may maintain its own binding activity, in which case the PDZ scaffold portion of a peptide ligand-grafted PDZ scaffold may mediate binding of the chimeric protein to a first target molecule. The grafted peptide ligand may mediate binding of the chimeric protein to a second target molecule”.

In some embodiments, the PDZ scaffold has a single binding site in a groove between the long a-helix and the beta strand (oriented parallel to the long a-helix), (see Figure 2 of Lee et a!., Cell Communication and Signalling 20108:8) with a highly conserved carboxylate-binding loop (R/K-xxx-G- t>-G- t> motif), where x is any amino acid residue and F is hydrophobic residues) located before the b strand. The first Gly residue in this motif is variable among canonical PDZ domains, and can be replaced by a Ser, Thr, or Phe residue. The second and the fourth residues are hydrophobic, such as Val, lie, Leu, or Phe. The side chains of both of these residues create the hydrophobic binding pocket of canonical PDZ domains. The third Gly residue in the loop region is fully conserved and is important for the formation of binding pocket. A single mutation of conserved residues of the motif may the binding affinity of the PDZ scaffolds to its binding partners. In some embodiments, the R/K-xxx-G- t>-G- t> motif of the PDZ scaffold maybe mutated to reduce or remove the native binding affinity.

Examples of grafted PDZ scaffolds according to the fifth aspect of the invention are shown in Figure 26C and Tables 23 and 25.

For example, a grafted PDZ scaffold may comprise a PDZ scaffold with the amino acid sequence of residues 1 to 108 of SEQ ID NO: 727 (PPX98 of Table 25 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first loop (loop 1 ; residues 28 to 30 of SEQ ID NO: 727), second loop (loop 2; residues 48 to 51 of SEQ ID NO: 727), or third loop (loop 3; residues 90 to 91 of SEQ ID NO: 727), or the first helix (helix 1 ; residues 70 to 80 of SEQ ID NO: 727) of the PDZ scaffold. Preferably, the peptide ligand is located in the second or third loop or the first helix.

Preferably, the E3 ligase-binding peptide ligand is located in the first loop. For example, the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop; the target-binding peptide ligand may be in the third loop and the E3 ligase-binding peptide ligand may be in the first loop; or the target-binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the first loop. Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof, peptides from BCL9, such as LxTLxxlQ, and SLxxlxxML and variants thereof, and helical beta-catenin binding sequences from the protein AXIN, such as ILxxHV and AxxILDxHV and variants thereof and variants thereof; and KRAS binding ligands, such as peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof, alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE or IxxTNxxKTxE and variants thereof; and peptides from RBP, such as

SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPLA or HYPWFKARLYPL and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG and GLDPETGELL and variants thereof.

In some embodiments, a grafted PDZ scaffold of the fifth aspect may comprise an amino acid sequence shown in Table 25 (SEQ ID NOs: 723-731) or a variant of an amino acid sequence shown in Table 25. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 25 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the PDZ scaffold sequence in a reference amino acid sequence of Table 25 is replaced by a different PDZ scaffold sequence. Suitable PDZ scaffold sequences are described above.

(vi) Ubiquitin or Ubiquitin-like Scaffolds

A sixth aspect relates to chimeric proteins that comprise Ubiquitin scaffolds (i.e. grafted Ubiquitin scaffolds). One or more peptide ligands are located in the Ubiquitin scaffold of the chimeric protein, at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305; or at positions between residues 25 to 26 or between residues 31 to 40 of SEQ ID NO: 737; or at positions between residues 13 to 14; between residues 22 to 23 or between residues 52 to 53 of SEQ ID NO: 751.

Ubiquitin is a small (8.6 kDa) globular, regulatory protein found in most tissues of eukaryotic organisms, Four genes in the human genome code for ubiquitin: UBB, UBC, UBA52 and RPS27A. The last four C-terminal residues (Leu-Arg-Gly-Gly) of the Ubiquitin domain extend from the compact structure to form a‘tail’. This tail plays an important role in the covalent conjugation of ubiquitin to target proteins by an isopeptide linkage between the C-terminal glycine and the epsilon amino group of lysine residues in the target proteins.

Ubiquitin performs its myriad functions through conjugation to a large range of target proteins. Ubiquitin domain is well known and well-characterized in the prior art. Ubiquitin domains are among the most extensively studied protein systems due to their ability to ubiquitinate and thereby mark proteins for degradation via the proteasome.

The invention adopts the well understood sequence-structure relationships of Ubiquitin domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

A Ubiquitin scaffold is a small, soluble, highly stable, single-domain folded protein, composed of a single long a helix, a short three and a half alpha-helix, and 4 b-strands comprising two b turns. Ubiquitin comprises an extraordinarily compact a/b structure (J. Mol. Biol., 1987 Apr. 5; 194(3):531-44) with almost 87% of the polypeptide chain is involved in the formation of the secondary structural elements by means of hydrogen bonds. Ubiquitin scaffolds may comprise marked hydrophobic region in the protein interior between the alpha helix and the b sheet. The alpha helix of the Ubiquitin scaffold is three turns in length and amenable for grafting peptide ligands.

A Ubiquitin scaffold has a length in the amino acid range of 1-76, 1-50, 1-105, and any amino acid length there between. A Ubiquitin scaffold is typically 8600 Da in MW, and such scaffolds are in the MW range of 8600, 5700, 11 850 Da.

A representative Ubiquitin scaffold, as used herein, may be 76 amino acids in length.

Figure 28 depicts a representative Ubiquitin scaffold. (PDB code: 2lj5)

A Ubiquitin scaffold possesses unique features with respect to protein characteristics, production and safety aspects. A Ubiquitin scaffold has favorable biochemical properties, e.g., a stable structure over a wide pH range, elevated temperatures and resistant to proteolytic degradation. A Ubiquitin scaffold which is derived from a human serum protein such as ubiquitin will exhibit low immunogenic potential when used in humans. The sequence of a Ubiquitin scaffold is fully conserved in mammals (FEBS Open Bio. 2015 Jul 10;5:579-93).

Suitable Ubiquitin scaffolds may include the Ubiquitin and Ubiquitin-like domains selected from the following proteins, without limitation: Ubiquitin-60S ribosomal protein L40 (amino acids 1-76; UniProtKB - P62987 (RL40JHUMAN); PDB: 2LJ5), Ubiquitin-40S ribosomal protein S27a (amino acids 1-76; P62979 (RS27A_HUMAN) ; PDB: 2KOX), Polyubiquitin-C (amino acids 1-76, 77-152, 153-228, 229-304, 305-380, 381- 456, 457-532, 533-608, 609- 684; UniProtKB - P0CG48 (UBC_HUMAN); PDB: 1C3T), Ubiquilin-1 (amino acids 37-111 ; UniProtKB - Q9UMX0 (UBQL1 JHUMAN) ; PDB: 2KLC), small ubiquitin-related modifier-1 (amino acids 20-97; UniProtKB - P63165 (SUM01 JHUMAN); PDB: 1A5R), E3 ubiquitin- protein ligase parkin (amino acids 1-76; UniProtKB - 060260 (PRKNJHUMAN); PDB: 1 IYF), ElonginB (amino acids 1-66; UniProtKB - Q15370 (ELOBJHUMAN), PDB: 2JZ3), BAG family molecular chaperone regulator 1 ( amino acids 144-224; UniProtKB - Q99933

(BAG1_HUMAN) , PDB: 1WXV), S3A1 , splicing factor 3 subunit 1 (amino acids 707-793; UniProtKB - Q15459 (SF3A1_HUMAN); PDB: 1ZKH) ; NEDD8 (amino acid 1-76; UniProtKB - Q 15843 (NEDD8_HUMAN); PDB: 1 NDD), E3 ubiquitin-protein ligase UHRF1 (amino acids 1-78; UniProtKB - Q96T88 (UHRF1 JHUMAN); PDB: 2FAZ), homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein (amino acids 10-72; UniProtKB - Q15011 (HERP1 JHUMAN), PDB: 1WGD), Large proline-rich protein BAG6 (amino acids 1-76; UniProtKB - P46379 (BAG6 JHUMAN); PDB: 4EEW); Ubiquilin-2 (amino acids 33-107; UniProtKB - Q9UHD9 (UBQL2_HUMAN), PDB: 1J8C); UV excision repair protein RAD23 homolog B (amino acids 1-79; UniProtKB - P54727 (RD23B_HUMAN); PDB: 1 P1A).

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable Ubiquitin scaffolds and it may include without any limitation: 1 UBI, 2MI8, 2MLB, 3VDZ, 5BVT, 1 P1A; 1JC8, 4EEW, 1 IYF etc.

Representative Ubiquitin Scaffolds Sequences

A Ubiquitin scaffold may comprise the amino acid sequence of SEQ ID NO: 291 or a variant thereof.

The above Ubiquitin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO:

292 or a variant thereof.

Rad23 functions as a natural substrate‘shuttle’ by binding simultaneously to ubiqutinated substrates (via its UIM domain) and to proteasome receptors (via its Ubl domain). In some embodiments, the Ubl domain of Rad23 may be used a representative Ubiquitin scaffold. In some embodiments the Ubiquitin scaffold derived from Ubl domain of Rad23 would be used to create a grafted Ubiquitin scaffold comprising one or more peptide ligand. Such a grafted Ubiquitin scaffold derived from Ubl domain of Rad23 may retain its natural ability to bind to proteasome receptors and may function to create a‘shuttle’ to deliver the target bound to the peptide ligand of the Ubiquitin grafted scaffold directly to the proteasome for degradation. In some embodiments, the Ubl domain of Rad23 is mutated to remove lysines and is preferable to use lysine free ubiquitin scaffolds for direct proteasome targeting.

Bag6 Ubl domain binds to the E3 ligase RNF26 and also has been shown to bind to the Rpn10 substrate receptor of the proteasome. In some embodiments, the Ubl domain of Bag6 may be used as a representative Ubiquitin scaffold. In some embodiments the

Ubiquitin scaffold derived from Ubl domain of Bag6 would be used to create a grafted Ubiquitin scaffold comprising one or more peptide ligand. Such a grafted Ubiquitin scaffold derived from Ubl domain of Bag6 may retain its natural ability to bind to E3 ligase and may function to create a‘shuttle’ to deliver the target bound to the peptide ligand of the Ubiquitin grafted scaffold directly to the proteasome for degradation. In some embodiments, the Ubl domain of Bag 6 is mutated to remove lysines and is preferable to use lysine free ubiquitin scaffolds for direct proteasome targeting.

Parkin Ub domain binds to the Rpn13 substrate receptor of the proteasome. In some embodiments, the Parking Ub domain may be used as a representative Ubiquitin scaffold. In some embodiments the Ubiquitin scaffold derived from Parkin Ub domain would be used to create a grafted Ubiquitin scaffold comprising one or more peptide ligand. Such a grafted Ubiquitin scaffold derived from Parkin Ub domain may retain its natural ability to bind to Rpn13 substrate and may function to create a‘shuttle’ to deliver the target bound to the peptide ligand of the Ubiquitin grafted scaffold directly to the proteasome for degradation. In some embodiments, the Ubl domain of Parkin Ub domain is mutated to remove lysines and is preferable to use lysine free ubiquitin scaffolds for direct proteasome targeting.

In some embodiments, the Ubiquitin scaffold of the invention is a naturally occurring frameshift variant that has a 19-residue unstructured C-terminal tail. In some embodiments, the Ubiquitin scaffold comprising a 19-residue unstructured C-terminal tail is capable of binding to substrate receptors of the proteasome. In some embodiments, the long unstructured tail may allow it to position its bound target close to the entrance cavity of the proteasome thereby facilitating the degradation process. In some embodiments, the Ubiquitin scaffold comprising a 19-residue unstructured C-terminal tail is mutated to remove lysines and is preferable to use lysine free ubiquitin scaffolds for direct proteasome targeting.

Table 27 shows the consensus sequence from the SMART database of domain

characteristic of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO

[(PUBM ED: 17491593), (PUBMED: 15479240)] and Nedd8 [(PUBMED:9857030)].

Table 28 shows the multiple alignment of SEQ ID NO: 291 to SEQ ID NO: 301. The beta- strands are boxed with a solid line, the alpha helices are boxed with a dashed line. The unboxed areas indicate loop or disordered regions.

Suitable Ubiquitin scaffolds include the Ubiquitin domains highlighted in Tables 27 and 28 (SEQ ID NOs: 291 , 293, 295, 297, 299, 301 , 303 and 305) or variants thereof. Other suitable Ubiquitin scaffolds include the Ubiquitin domains of residues 1 to 86 of SEQ ID NO: 737 or residues 1 to 88 of SEQ ID NO: 751.

Suitable positions for peptide ligand insertions or replacements for the Ubiquitin scaffolds for variants of sequence listed in Tables 27 and 28 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID NOs: 291 ,

293, 295, 297, 299, 301 , 303, 305, 737 and/or 751.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NO: 291 , 293, 295, 297, 299, 301 , 303 and/or 305.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 25 to 26; and/or between residues 31 to 40 of SEQ ID NO: 737.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 13 to 14; between residues 22 to 23; and/or between residues 52 to 53; of SEQ ID NO: 751.

Preferred Ubiquitin scaffolds lack lysine residues, for example, to avoid unwanted

ubiquitination. For example, the lysine residues in a Ubiquitin domain may be replaced by E, N, Q , S or R to generate a Ubiquitin scaffold. A suitable lysine-free Ubiquitin scaffold may comprise the amino acid sequence of SEQ ID NO: 291 or a variant thereof. The Ubiquitin domain has seven Lys residues (Lys 6, Lys 11 , Lys 27, Lys 29, Lys 33, Lys 48 and Lys 63). In some embodiments, residue Lys 6, Lys 27, Lys 29 and Lys 48 of a Ubiquitin scaffold is replaced with arginine. In some embodiments, residue Lys11 of a Ubiquitin scaffold is replaced with a polar or small amino acid such as asparagine. In some embodiments, residue Lys 33 of a Ubiquitin scaffold is replaced with glutamine. In some embodiments, residue Lys 62 is replaced with threonine.

In some embodiments, all lysines are replaced with a polar amino residue selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, asparagine, glutamine and histidine. In some embodiments, all lysines are replaced with arginines. In the above embodiments, any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold. A lysine-attenuated Ubiquitin scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids, may comprise the amino acid sequence of SEQ ID NO: 305 or a variant thereof.

In some embodiments, a lysine-free Ubiquitin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 306 or a variant thereof.

The modelled grafting of the Ubiquitin scaffold is based on SEQ ID NO: 291 and the PDB file: 2lj5. Figure 29 show a representative example of a grafted Ubiquitin scaffold with peptide ligands grafted on to them, indicated by shaded loops. A threading program such as SwissModeller (Nucleic Acids Res. 46(W1), W296-W303 (2018)) and an ab initio folding program such as l_tasser (Protein structure and function prediction. Nature Methods, 12: 7-8 (2015)) or Robetta (Nucleic Acids Research, Vol. 32, No. S2. (2004), pp. W526-W531) may be used to create models of grafted scaffolds.

The b-1 strand of a Ubiquitin scaffold is located within the positions corresponding to residues 1 to 7 of SEQ ID NO: 291 and SEQ ID NO: 305. The b-2 strand of a Ubiquitin scaffold is located within the positions corresponding to residues 11 to 17 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The b-3 strand of a Ubiquitin scaffold is located within the positions corresponding to residues 40 to 45 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The b-4 strand of a Ubiquitin scaffold is located within the positions corresponding to residues 48 to 50 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The b-5 strand of a Ubiquitin scaffold is located within the positions corresponding to residues 65 to 72 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The long a-helix of a Ubiquitin scaffold is located within the positions corresponding to residues 23 to 33 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The short a-helix of a Ubiquitin scaffold is located within the positions corresponding to residues 57 to 58 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The disordered regions or the loop regions of a Ubiquitin scaffold are located within the positions corresponding to residues 8 to 9 for loop 1 , 51 to 56 for loop 2 and 59 to 64 for loop 3 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The first loop of a Ubiquitin scaffold is located within the positions corresponding to residues 8 to 9 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The second loop of a Ubiquitin scaffold is located within the positions corresponding to residues 51 to 56 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

The second loop of a Ubiquitin scaffold is located within the positions corresponding to residues 51 to 56 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

Grafted Ubiquitin Scaffolds

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 8 and 9 of loop 1 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 53 and 54 of loop 2 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 62 and 63 of loop 3 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 25 to 26 of loop 1 of SEQ ID NO: 737.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 13 to 14 of loop 1 of SEQ ID NO: 751.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 22 to 23 of loop 2 of SEQ ID NO: 751.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand within a loop (a“loop peptide”) for example inserted between residues 52 to 53 of loop 3 of SEQ ID NO: 751.

In some embodiments, a grafted Ubiquitin scaffold comprises a peptide ligand grafted onto a helix (a“helix peptide”), e.g. residues 23 and 33 of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305 or residues 31 to 40 of SEQ ID NO: 737.

In some embodiments, a grafted Ubiquitin scaffold comprises a first peptide ligand between positions for example between 8 and 9 in loop 1 of the Ubiquitin scaffold SEQ ID NO: 291 and 305. In some embodiments, a grafted Ubiquitin scaffold comprises a second peptide ligand for example inserted in loop 2 between the positions 53 and 54 of the Ubiquitin scaffold.

In some embodiments, a grafted Ubiquitin scaffold comprises a first peptide ligand between positions for example between residues 25 and 26 in loop 1 or between residues 31 and 40 in helix 1 of the Ubiquitin scaffold of SEQ ID NO: 737. In some embodiments, a grafted Ubiquitin scaffold comprises a first peptide ligand at a position for example between residues 13 to 14 in loop 1 or between residues 22 to 23 in loop 2 of the Ubiquitin scaffold of SEQ ID NO: 751. In some embodiments, a grafted

Ubiquitin scaffold comprises a second peptide ligand for example inserted in loop 3 between the positions 52 to 53 of the Ubiquitin scaffold of SEQ ID NO: 751.

In some embodiments, a grafted Ubiquitin scaffold comprises a first peptide ligand between positions for example between 25 and 26 in loop 1 of the Ubiquitin scaffold SEQ ID NO: 737. In some embodiments, a grafted Ubiquitin scaffold comprises a second peptide ligand for example inserted in loop 2 between the positions 53 and 54 of the Ubiquitin scaffold.

In some embodiments, a grafted Ubiquitin scaffold comprises a first loop peptide ligand inserted between positions 8 and 9 of the Ubiquitin scaffold and a third loop peptide ligand inserted between the positions 62 and 63 of the Ubiquitin scaffold SEQ ID NO: 291 and 305. In some embodiments, a grafted Ubiquitin scaffold comprises a first helical peptide grafted onto the helix between positions 23 and 33 of the Ubiquitin scaffold and a second loop peptide ligand inserted between the positions 8 and 9 in loop 1 of the Ubiquitin scaffold SEQ ID NO: 291 and 305.

In some embodiments, a grafted Ubiquitin scaffold comprises a first helical peptide grafted onto the helix between positions 23 and 33 of the Ubiquitin scaffold and a second loop peptide ligand inserted between the positions 53 and 54 in loop 2 of the Ubiquitin scaffold SEQ ID NO: 291 and 305.

In some embodiments, a grafted Ubiquitin scaffold comprises a first helical peptide grafted onto the helix between positions 23 and 33 of the Ubiquitin scaffold and a third loop peptide ligand inserted between the positions 62 and 63 of the Ubiquitin scaffold.

In some embodiments, a grafted Ubiquitin scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted Ubiquitin scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e., a target protein and an E3 ligase. In some embodiments, a grafted Ubiquitin scaffold is created by insertion at the position of solvent exposed residues of a loop (for example, inserted between the position of residues 53 and 54 or residues 62 and 63 of SEQ ID NO: 291 and 305) with solvent facing residues of the loop peptide ligand.

In some embodiments, a grafted Ubiquitin scaffold is created by isomorphic replacement of those residues E24, N25, A28, K29, Q31 , D32 and K33 of a helix which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.

A grafted Ubiquitin scaffold comprises a loop peptide ligand in a loop portion of the Ubiquitin scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). In some embodiments, a grafted Ubiquitin scaffold comprises a loop peptide ligand inserted in to a first loop that starts on residue number 8 of the Ubiquitin scaffold. Loops in a Ubiquitin domain generally show a high occurrence of insertions across the HMM logo. The loops connecting beta-strands are ideal places for loop grafting of peptide ligands because the loop regions do not have any conserved residues.

In some embodiments, any residues of the Ubiquitin scaffold may be replaced with residues of peptide ligands except for the positions of the Ubiquitin scaffold marked by residues: I23, V26, K27, and 130

A grafted Ubiquitin scaffold comprises a helical peptide ligand in a helical portion of the Ubiquitin scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). The long helix in a Ubiquitin scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.

A grafted Ubiquitin scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the Ubiquitin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 8 of SEQ ID NO: 291 or 305. A loop peptide ligand may be inserted into the Ubiquitin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 9 of SEQ ID NO: 291 or 305.

Examples of grafted ubiquitin scaffolds according to the sixth aspect of the invention are shown in Figure 31 B and Tables 26 and 29.

In some embodiments, a grafted ubiquitin scaffold may comprise a ubiquitin scaffold with the amino acid sequence of residues 1 to 86 of SEQ ID NO: 737 (PPX6 of Table 29 without the HA Tag). In some embodiments, a grafted ubiquitin scaffold may comprise a proteasome binding ligand. The proteasome binding ligand may be endogenous to the ubiquitin scaffold (e.g. hPLIC2). The proteasome binding ligand may comprise the sequence IXA(X) ₂ I H , where x at each position is independently any amino acid. For example a proteasome binding ligand may be located at residues 51 to 75 of SEQ ID NO: 737. A target-binding peptide ligand may be grafted into any of the first, second, and third loops and the first helix of the ubiquitin scaffold. Preferably, the peptide ligand is located in the first loop (loop 1 residues 25 to 26 of SEQ ID NO: 737) or the first helix (helix 1 ; residues 31 to 40 of SEQ ID NO: 737). In embodiments in which the grafted ubiquitin scaffold comprises a proteasome binding ligand, it may lack an E3 ligase binding ligand.

In other embodiments, a grafted ubiquitin scaffold may comprise a grafted ubiquitin scaffold may comprise a ubiquitin scaffold with the amino acid sequence of residues 1 to 88 of SEQ ID NO: 751 (PX112 of Table 29 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first loop (loop 1 ;

residues 13 to 14 of SEQ ID NO: 751), second loop (loop 2; residues 22 to 23 of SEQ ID NO: 751), and third loop (loop 3; residues 52 to 53 of SEQ ID NO: 751) of the ubiquitin scaffold. Preferably, the peptide ligand is located in the first or second loop. Preferably, the E3 ligase-binding peptide ligand is located in the third loop. For example, the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the third loop; the target-binding peptide ligand may be in the second loop and the E3 ligase- binding peptide ligand may be in the third loop. In some embodiments, the E3 ligase-binding peptide ligand may be a KRAS binding sequence that is endogenous to the ubiquitin scaffold (e.g. Raf-RBD).

Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS, GSEELEALEALELDEAS and GSEELEALEALELDEASGS, peptides from BCL9, such as TLxxlQ, LxTLxxlQ, and SLxxlxxML, and helical beta-catenin binding sequences from the protein AXIN, such as ILxxHV and AxxILDxHV; and KRAS binding ligands, such as peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS or HYPWFKARLYPL and KRAS binding ligands, such as alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as LTNxLK, TNxxKxxE or IxxTNxxKTxE; peptides from KBL, such as PLYISY and PLYISYPV, and peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and HYPWFKARLYPL. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG and GLDPETGELL and SCF ^Sk P ² binding sequences from p27, such as AGSNEQEPNR and AGSNEQEPKKA. Suitable proteasome- binding peptide ligands include IxA.

In some embodiments, a grafted ubiquitin scaffold of the sixth aspect may comprise an amino acid sequence shown in Table 29 (SEQ ID NOs: 732 to 751) or a variant of an amino acid sequence shown in Table 29. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 29 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the ubiquitin scaffold sequence in a reference amino acid sequence of Table 29 is replaced by a different ubiquitin scaffold sequence. Suitable ubiquitin scaffold sequences are described above.

Figure 30 shows a model of ubiquitin scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, Affibody scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl

The Ubiquitin scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Ubiquitin scaffolds.

(vii) GB1 Scaffolds

A seventh aspect relates to chimeric proteins that comprise GB1 scaffolds (i.e. grafted GB1 scaffolds). One or more peptide ligands are located in the GB1 scaffold of the chimeric protein, for example at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID Nos: 307, 309, 311 and 313-348 or between residues 28 to 36, residues 39 to 40 or residues 49 to 50 of the scaffold.

GB1 (Immunoglobulin-binding domain B1 of streptococcal protein G) domains are well known and well-characterized in the prior art. GB1 domains are among the most extensively used model systems in the area of protein folding and design (Park, et al. Biochemistry, 36:14277-14283 (1997); J Mol Biol. 2003 Oct 10;333(1): 141-52; FEBS Letters 398 (1996) 312-316)).

The invention adopts the well understood sequence-structure relationships of GB1 domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

A GB1 scaffold is a small, soluble, stable, single-domain folded protein, composed of a single long a-helix and a 4 stranded b-sheet containing two b-turns. A GB1 scaffold, as used herein, has a length in the amino acid range of 50-80, and any amino acid length there between. A GB1 scaffold is typically 6241 daltons in MW, and such scaffolds are in the MW range of 5500-9000 daltons.

A representative GB1 domain scaffold may be about 58 amino acids in length. Figure 33 is a schematic of the structure of a representative GB1 scaffold. A GB1 scaffold comprises no disulfide bonds or free cysteines or metal binding sites and has a hydrophobic core formed by the four beta sheets and one alpha helix. The central part of the hydrophobic core of a GB1 scaffold comprises residues Leu-5, Leu-7, Ala-26, Phe-30, Ala34, Val-52 and Val-54 with residues Tyr-3, Gly-9, Tyr-33, and Val-39 at the boundaries. (Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11963-8).

Suitable GB1 scaffolds may include a GB1 domain selected from the following proteins, without limitation: Immunoglobulin G-binding protein G (UniProtKB - P06654

(SPG1_STRSG) amino acids 228-282, 298-352); IgG binding protein Zag (UniProtKB - B4U242 (B4U242_STREM) amino acids 245-298, 316-370), (UniProtKB - A0A072EKT7 (A0A072EKT7_9STRE) amino acids 230-286), (UniProtKB - A0A380JRD4

(A0A380JRD4_9STRE) amino acids 233-286, 304-358); Mag protein (UniProtKB - A0A2D4DSS1 (A0A2D4DSS1_STRCB) amino acids 307-361), (UniProtKB - Q53974 (Q53974_STRDY) amino acids 258-312); Immunoglobulin G-binding protein ((UniProtKB - A0A2X3XQR1 (A0A2X3XQR1_STREQ), amino acids 224-278, 294-348, 364-418, 434- 488); (UniProtKB - A0A2Z6G3G4 (A0A2Z6G3G4_STRDY, amino acids 13-67, 86-137, 153- 207, 223-277), (UniProtKB - A0A380JVQ2 (A0A380JVQ2_STRDY) amino acids 276-330, 346-400, 416-470), (UniProtKB - M4YXE4 (M4YXE4_STREQ) amino acids 240-294, 310- 364), (UniProtKB - C5WHI8 (C5WHI8_STRDG) amino acids 303-357, 373-427); Mig (UniProtKB - Q93EM8 (Q93EM8_STRDY) amino acids 224-278, 294-348, 364-418, 434- 488, 504-558); Streptococcal surface protein (UniProtKB - Q53975 (Q53975_STRDY) amino acids 224-278, 294-348, 364-418, 434-488, 504-558), Protein G' (UniProtKB - Q54181 (Q54181_STRSG) amino acids 61-115, 131-185), Protein G IgG Fc binding domain

(UniProtKB - Q53337 (Q53337_9STRE) amino acids, Protein LG (UniProtKB - Q53291 (Q53291_FINMA) amino acids 330-384, 400-454), YSIRK family Gram-positive signal peptide (UniProtKB - K9E8M0 (K9E8M0_9LACT) amino acids); B domain protein

(UniProtKB - E4KPW8 (E4KPW8_9LACT) amino acids 43-97); CAP225 Scaffolded V1V2 ( UniProtKB - A0A3F2YM25 (A0A3F2YM25_9HIV1) amino acids 89-130).

Table 27 shows the alignment of GB1 domains in UniProt. The query sequence is shown in bold. A representative non-limiting example of sequences are shown below. The consensus sequence derived from a representative sample of GB1 domain family is shown. The amino acids in bold are conserved across all members of the family; those boxed are highly favoured. The positions of the structural elements of the GB1 domain are indicated.

Positions on the alpha-helix that can be modified for grafting of a binding motif are indicated with an X. Positions between which a loop can be grafted are indicated with **.

Suitable GB1 scaffolds include the GB1 domains shown in Tables 31 and 32 and SEQ ID NOs: 307, 309, 311 and 313-348 or variants thereof. Suitable GB1 scaffolds also include the GB1 domains of residues 1 to 64 of SEQ ID NO: 756 or variants thereof.

Suitable positions for peptide ligand insertions or replacements for the GB1 scaffolds for variants of sequence listed in Tables 31 and 32 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID NOs: 307,

309, 311 and 313-34.

In some embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313-34 of the GB1 scaffold.

In other embodiments, one or more peptide ligands may be inserted or replaced at positions between residues 28 to 36 or between residues 39 and 40 or between residues 49 and 50 of SEQ ID NO: 756 of the GB1 scaffold.

A GB1 scaffold may comprise the amino acid sequence of SEQ ID NO: 307 or a variant thereof.

where n is 0-30 and X and Z is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine, phospho-serine, phospho- threonine and phospho-tyrosine, acetylated amino acids.

The above GB1 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 308 or a variant thereof.

where n is 0-30 and Y and W is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 307. For instance, Y is selected from the group of codons“att, ate, and ata” if X of SEQ ID NO: 307 were to be Isoleucine. Similarly, Y is selected from the group of codons“ett, etc, eta, ctg, tta, ttg” if X of SEQ ID NO: 307 were to be Leucine. Table 8 provides codons for each amino acid that can be used to construct a suitable nucleic acid sequence that encodes variants of SEQ ID NO: 307.

Preferred GB1 scaffolds lack lysine residues, for example, to avoid ubiquitination of the scaffold. For example, the lysine residues in a GB1 domain may be replaced by E, N, Q, S or R to generate a GB1 scaffold. A suitable lysine-free GB1 scaffold may comprise the amino acid sequence of SEQ ID NO: 307 or a variant thereof.

In some embodiments, residue Lys4 of a GB1 scaffold is replaced with arginine. In some embodiments, residue Lys9 of a GB1 scaffold is replaced with asparagine. In some embodiments, residue Lys13 of a GB1 scaffold is replaced with serine. In some

embodiments, residue Lys28 of a GB1 scaffold is replaced with glutamine. In some embodiments, residue Lys31 is optionally replaced with arginine. In some embodiments, residue Lys50 is optionally replaced with a polar amino residue selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, asparagine, glutamine and histidine. In the above embodiments, any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold. A lysine-attenuated GB1 scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids, may comprise the amino acid sequence of SEQ ID NO: 309 or a variant thereof.

(SEQ ID NO: 309; positions of replaced K residues in bold and underlined) where n is 0-30, and X and Z is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.

In some embodiments, a lysine-free GB1 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 310 or a variant thereof.

where n is 0-30 and Y and W is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 309. For instance, Y is selected from the group of codons“att, ate, and ata” if X of SEQ ID NO: 309 were to be Isoleucine. Similarly Y is selected from the group of codons“ett, etc, eta, ctg, tta, ttg” if X of SEQ ID NO: 309 were to be Leucine. The aforesaid codon table provides codons for each amino acid that can be used to construct a suitable nucleic acid sequence that encodes variants of SEQ ID NO:

309.

Preferred GB1 scaffolds have a high thermostability, for example, to avoid denatu ration to an unfolded inactive state. Stabilising mutants have been identified. For example, the following amino acids resulted in large increases in thermostability, 3F, 7T, 161, 181, 25E, 291, 391 (Malakauskas SM & Mayo SL, Design, structure and stability of a hyperthermophilic protein variant, (1998), Nat. Struct. Biol., 5: 470-475). SEQ ID NO: 311 is a lysine-free GB1 scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids and further replacements have been made to improve thermostability or a variant thereof.

where n is 0-30 and X and Z is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.

where n is 0-30 and Y and W is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 311. For instance, Y is selected from the group of codons“att, ate, and ata” if X of SEQ ID NO: 311 were to be Isoleucine. Similarly Y is selected from the group of codons“ett, etc, eta, ctg, tta, ttg” if X of SEQ ID NO: 311 were to be Leucine. The aforesaid codon table (Table 8) provides codons for each amino acid that can be used to construct a suitable nucleic acid sequence that encodes variants of SEQ ID NO: 311.

The model of grafting originated from PDB code 2PLP and numbering is based on the structure derived from 2PLP and also that given in Table 32.

The b-1 strand of a GB1 scaffold is located within the positions corresponding to residues 2 to 8 of SEQ ID NOs: 307, 309, 311 and 313-348.

The b-2 strand of a GB1 scaffold is located within the positions corresponding to residues (11 + Xn) to (17 + Xn) of SEQ ID NOs: 307, 309, 311 and 313-348.

The b-3 strand of a GB1 scaffold is located within the positions corresponding to residues (41 + Xn) to (45 + Xn) of SEQ ID NOs: 307, 309, 311 and 313-348.

The b-4 strand of a GB1 scaffold is located within the positions corresponding to residues (50 + Xn + Zn) to (55 + Xn + Zn) of SEQ ID NOs: 307, 309, 311 and 313-348.

The long a-helix of a GB1 scaffold is located within the positions corresponding to residues (22 + Xn) to (35 + Xn) of SEQ ID NOs: 307, 309, 311 and 313-348.

The disordered regions or the loop regions of a GB1 scaffold are located within the positions corresponding to residues (9 to (10 + Xn)), ((18+ Xn) to (21 + Xn )), ((36+ Xn) to (40 + Xn )), ((46+ Xn) to (49 + Xn + Zn )) of SEQ ID NOs: 307, 309, 311 and 313-348.

The first loop of the GB1 scaffold is at a position corresponding to residues 9 to 10 of SEQ ID NOs: 307, 309, 311 and 313-348

The second loop of the GB1 scaffold is at a position corresponding to residues 18 to 21 of SEQ ID NOs: 307, 309, 311 and 313-348.

The third loop of the GB1 scaffold is at a position corresponding to residues 36 to 40 of SEQ ID NOs: 307, 309, 311 and 313-348.

The fourth loop of the GB1 scaffold is at a position corresponding to residues 46 to 49 of SEQ ID NOs: 307, 309, 311 and 313-348.

In some embodiments, a grafted GB1 scaffold comprises a peptide ligand within a loop (a “loop peptide”) (between residues 9-10 or 46-49 of SEQ ID NOs: 307, 309, 311 and 313- 348). In some embodiments, a grafted GB1 scaffold comprises a peptide ligand within a helix (a“helix peptide”) (residues 22 to 35).

In some embodiments, a grafted GB1 scaffold comprises a first loop peptide between positions 9 and 10 of the GB1 scaffold and a second loop peptide between the positions 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313-348 of the GB1 scaffold.

In some embodiments, a grafted GB1 scaffold comprises a first loop peptide between positions 28 and 36 of the GB1 scaffold and a second loop peptide between the positions 39 and 40 of SEQ ID NOs: 756 of the GB1 scaffold.

In some embodiments, a grafted GB1 scaffold comprises a first loop peptide between positions 39 and 40 of the GB1 scaffold and a second loop peptide between the positions 49 and 50 of SEQ ID NOs: 756 of the GB1 scaffold.

In some embodiments, a grafted GB1 scaffold comprises a first helical peptide between positions 22 and 35 of SEQ ID NOs: 307, 309, 311 and 313-348 of the GB1 scaffold and a second loop peptide between the positions 9 and 10 of SEQ ID NOs: 307, 309, 311 and 313-348 of the GB1 scaffold.

In some embodiments, a grafted GB1 scaffold comprises a first helical peptide between positions 22 and 35 of SEQ ID NOs: 307, 309, 311 and 313-348 of the GB1 scaffold and a second loop peptide ligand between the positions 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313-348 of the GB1 scaffold.

In some embodiments, a grafted GB1 scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and orientations such that the two peptide ligands do not come into contact with each other. It is preferred that a grafted GB1 scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e. , a target protein and an E3 ligase.

In some embodiments, a grafted GB1 scaffold is created by isomorphic replacement of solvent-exposed residues of a loop 9 to 10 and/or 46 to 49 of SEQ ID NOs: 307, 309, 311 and 313-348) with solvent facing residues of the loop peptide ligand.

In some embodiments, a grafted GB1 scaffold comprises isomorphic replacement of those residues 23, 24, 26, 27, 28, 34 and 35 of a helix which are solvent-exposed as indicated in Table 32 and not buried into the hydrophobic core of the scaffold with solvent-facing residues of the helix peptide ligand.

A grafted GB1 scaffold comprises a loop peptide ligand in a loop portion of the GB1 scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a GB1 domain generally show a high occurrence of insertions across the HMM logo. The loops connecting beta-strands are ideal sites for loop grafting of peptide ligands because the loop regions do not have any conserved residues.

A grafted GB1 scaffold comprises a helical peptide ligand in a helical portion of the GB1 scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). The long helix in a GB1 scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.

A grafted GB1 scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the GB1 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 1 or 9 or 46, 47, 48 or 55 of SEQ ID NOs: 307, 309, 311 and 313-348. A loop peptide ligand may be inserted into the GB1 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 2 or 10 or 46, 47, 48 of SEQ ID NOs: 307, 309, 311 and 313-348.

A grafted GB1 scaffold also may contain a peptide ligand within a helical region of the scaffold (i.e. , a“helical peptide” is a peptide which is positioned in a helical structure of the scaffold). In some embodiments, a grafted GB1 scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C-terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 55 of SEQ ID NO: NO: 307, 309 or 311. In some embodiments, a grafted GB1 scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 1 of SEQ ID NO: NO: 307, 309 or 311.

A schematic model of the grafted GB1 scaffold is shown in Figure 33. The helix of the GB1 scaffold that can be modified to provide a novel binding function is shown. An example of a loop insertion is shown, the alternative position for a loop insertion is indicated.

Figure 35 shows is an example of a GB1 scaffold with a first grafted peptide ligand, which is a loop, and a second grafted peptide ligand, which is a helix. The Figure shows two different views of the modelled structure of the complex of loop-helix-grafted GB1 domain (light grey) in complex with KRAS (dark grey) and the E2-E3 Cullin-Keapl complex (shades of grey). The figure 35 shows a model of GB1 scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex. The Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between KRAS, SOS peptide, GB1 scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl .

In some embodiments, the GB1 scaffold has a native binding affinity to IgG (Fc). The binding interaction between the GB1 scaffold and IgG (Fc) is mediated by the amino acid residues located on the solvent-exposed face of the alpha helix, namely residue positions Glu(E)26, Lys(K)30 and Asn(N) 35. A single mutation of Glu(E)26 position has shown to reduce the binding affinity of the GB1 to IgG (Fc) by 10,000 fold. (Sloan DJ & Hellinga HW Prot Sci (1999) 8:1643-1648). In some embodiments, the position Glu(E)26 of the GB1 scaffold maybe mutated to reduce or remove the binding affinity to IgG (Fc). In some embodiments, all the residues Glu(E)26, Lys(K)30 and Asn(N) 35 are replaced with residues of the peptide ligand to provide a new biological molecule (degron or substrate) thereby abolishing the native binding affinity to the IgG (Fc) protein.

Examples of grafted GB1 scaffolds according to the seventh aspect of the invention are shown in Figure 36B and Tables 30 and 33 (SEQ ID NOs 752-760).

For example, a grafted GB1 scaffold may comprise a trefoil scaffold with the amino acid sequence of residues 1 to 64 of SEQ ID NO: 756 (PPX117 of Table 33 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first, second, and third loops of the GB1 scaffold. Preferably, the peptide ligand is located in the first or second loop or the helical region. Preferably, the E3 ligase- binding peptide ligand is located in the first, second or third loop. For example, the target binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop; the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop; or the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the third loop.

Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof, peptides from BCL9, such as LAEIIFRLYAA, TLxxlQxxL, LXTLxxlQ, and SLxxlxxML and variants thereof, and KRAS binding ligands, for example sequences from the protein SOS1 (Son of sevenless homolog 1), such as IAETNFRKYAE, LTNXLK, TNxxKxxE or IxxTNxxKTXE; peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and

HYPWFKARLYPL and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, GGLDPETGELL and GLDPETGELL and variants thereof and SCF ^Skp2 binding sequences from p27, such as FIFRWYA, AGSNEQEPNR and variants thereof.

In some embodiments, a grafted GB1 scaffold of the seventh aspect may comprise an amino acid sequence shown in Table 33 or a variant of an amino acid sequence shown in Table 33. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 33 are replaced by a different peptide ligand.

Suitable peptide ligands are described below. Variants may also include variants in which the GB1 scaffold sequence in a reference amino acid sequence of Table 33 (SEQ ID NOs 752-759)is replaced by a different GB1 scaffold sequence. Suitable GB1 scaffold sequences are described above.

(viii) VWV Scaffolds

An eighth aspect relates to chimeric proteins that comprise VWV scaffolds (i.e. grafted VWV scaffolds). One or more peptide ligands are located in the VWV scaffold of the chimeric protein, for example in the first or second loops, or in a helical region, if present (helixWW scaffold).

A VWV domain (also known as an rsp5-domain or VVWP repeating domain) is a common structural domain of approximately 40 amino-acids found in signalling proteins of bacteria, yeast, plants, viruses and animal protein (VWV refers to the presence of two conserved tryptophan residues in most VWV domains) (Staub et al (1996) Structure 4 5 495-499). VWV domains may mediate binding to proteins with particular proline-motifs, [AP]-P-P-[AP]-Y, and/or phosphoserine- phosphothreonine-containing motifs. The VWV domain is sufficiently small to be synthetically tractable (hence allowing incorporation of unnatural amino acids) as well as genetically encodable.

The structure of VWV domains has been described in the art (see for example Pfam

PF00397, PDB 1 E0M; Macias et al (2000) Nat Struct Mol Biol 7: 375; Macias (1996) Nature 382 646-649).

The invention adopts the well understood sequence-structure relationships of VWV domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a VWV scaffold is folded protein comprising a triple-stranded anti parallel beta-sheet (i.e. a VWV domain structure). In some embodiments, a VWV scaffold may have high cell penetrating abilities, owing to its smaller size.

A VWV scaffold, as used herein, has a length in the amino acid range of 25 to 65 amino acids, for example 27 to 61 amino acids, 30 to 60 amino acids, and any amino acid length there between.

A VWV scaffold may be in the MW range of 3520 to 6620 kDa, typically about 4700 kDa in

MW. A representative VWV scaffold is shown in Figure 38.

Suitable VWV scaffolds useful according to the invention include the VWV domains selected from the following, without limitation: dystrophin; Peptidyl-prolyl cis-trans isomerase (NIMA- interacting 1) (VWV domain 5-35 amino acids; UniProtKB - Q13526 (PIN1_HUMAN); PDB 1 I6C), Transcriptional coactivator YAP1 ( VWV domains 171-204 & 230-263 amino acids, UniProtKB - P46937 (YAP1JHUMAN), PDB: 1 K9R), VVWP3-1 ; VVWP3-2; E3 ubiquitin- protein ligase NEDD4-like (VWV domains 221-254, 414-447, 526-559, 577-610 amino acids; UniProtKB - Q8CFI0 (NED4L_MOUSE); PDB: 1WR4), Membrane-associated guanylate kinase, VWV and PDZ domain-containing protein 1 (VWV domains 300-333 & 359-362 amino acids, UniProtKB - Q96QZ7 (MAGI I _{_} HUMAN), PDB: 2YSE), Pre-mRNA-processing protein PRP40 (VWV domains 1-31 & 42-72 amino acids, UniProtKB - P33203

(PRP40_YEAST); PDB: 106W) , E3 ubiquitin-protein ligase Itchy homolog (VWV domains 326-359, 358-391 , 438-471 , 478-511 amino acids; UniProtKB - Q96J02 (ITCH_HUMAN) PDB: 2KK), BAG family molecular chaperone regulator 3 (VWV domains 20-54 and 124-154; UniProtKB - 095817 (BAG3_HUMAN)).

Other examples of suitable VWV scaffolds are shown in Table 34 and Table 35 and described for example in Otte et al (2003) Protein Science 12(3) 491-500. Other suitable VWV scaffolds may comprise a VWV domain consensus sequence shown in Tables 36 and 37. Other suitable VWV scaffolds may comprise a VWV domain of residues 1 to 44 of SEQ ID NO: 763.

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable VWV scaffolds useful according to the invention without any limitation: 1 E0M, 1 M8I, 1 I6C, 2E45, 2JX8, 2MDC, 2DK1 , 2JV4, 1 E0L, 1 E0N, 1 K9R, 1WMV, 1WR3, 1WR4, 1WR7, 1ZR7, 2IDH, 2JXW, 2M8J and 2N4R.

In some embodiments, a VWV scaffold may comprise the amino acid sequence of SEQ ID NO: 349 or a variant thereof.

(SEQ ID NO: 349 loop residues

underlined).

The VWV scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 350 or a variant thereof. In some embodiments, a VWV scaffold may comprise the amino acid sequence of SEQ ID NO: 351 or a variant thereof.

51 ; loop residues

underlined).

The VWV scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 352 or a variant thereof.

In some embodiments, a VWV scaffold may comprise the amino acid sequence of SEQ ID NO: 353 or a variant thereof.

NO: 353 the loop residues are underlined).

The VWV scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 354 or a variant thereof.

In some embodiments, a VWV scaffold may comprise the amino acid sequence of SEQ ID NO: 355 or a variant thereof.

SMGLPPGWDEYKTHNGKTYYYNHNTKTSTWTDPRMSS (SEQ ID NO: 355 the loop residues are underlined).

The VWV scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 356 or a variant thereof.

Preferred VWV scaffolds lack lysine residues, for example to avoid unwanted ubiquitination. For example, the lysine residues in a VWV domain may be replaced by a polar or charged amino acid to generate a VWV scaffold. The VWV domain of SEQ ID NO: 349 has two lysine residues (Lys1 and Lys8). In some embodiments, Lys1 of the VWV domain may be replaced with serine. In some embodiments, the residues Lys8 of the VWV domain may be replaced with glutamine. A suitable lysine-free VWV scaffold may comprise the amino acid sequence of SEQ ID NO: 357 or a variant thereof. positions of

replaced K residues underlined) A lysine- free WW scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 358 or a variant thereof.

The first loop of a VWV scaffold is located within the positions corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355 and residues 16 to 17 of SEQ ID NO: 763.

The second loop of a VWV scaffold is located within the positions corresponding to residues 23 to 25 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 29 to 31 of SEQ ID NO: 351 , residues 32 to 34 of SEQ ID NO: 353, residues 24 to 26 of SEQ ID NO: 355 and residues 26 to 27 of SEQ ID NO: 763.

Peptide ligands may be located in one or both of the first and second loops in the VWV scaffold. Preferably, a first peptide ligand is located in the first loop and a second peptide ligand is located in the second loop of the VWV scaffold.

A grafted VWV scaffold contains a peptide ligand in a loop portion of the VWV scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). The peptide ligand may be heterologous.

In some embodiments, a grafted VWV scaffold may contain a peptide ligand inserted within the first loop (a“loop peptide”) for example between residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355 and residues 16 to 17 of SEQ ID NO: 763.

For example, a peptide ligand may be located in the VWV scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of the residue corresponding to 11 , 12, 13 or 14 of SEQ ID NO: 349 or SEQ ID NO: 357, the residue correspond to residue 17, 18 or 19 of SEQ ID NO: 351 , the residue correspond to residue 23, 24 or 25 of SEQ ID NO: 353, or the residue correspond to residue 13, 14 or 15 of SEQ ID NO: 355 or the residue corresponding to 15 or 16 of SEQ ID NO: 763. The peptide ligand may be inserted into the VWV scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of the residue corresponding to 13, 14, 15 or 16 of SEQ ID NO: 349 or SEQ ID NO: 357, the residue correspond to residue 19, 20 or 21 of SEQ ID NO: 351 , the residue correspond to residue 25, 26 or 27 of SEQ ID NO: 353, the residue correspond to residue 15, 16 or 17 of SEQ ID NO: 355 or the residue correspond to residue 17 or 18 of SEQ ID NO: 355. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355 or residues 16 to 17 of SEQ ID NO: 763.

In some embodiments, a grafted VWV scaffold may contain a peptide ligand within the second loop (a“loop peptide”) for example between residues corresponding to residues 23 to 25 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 29 to 31 of SEQ ID NO: 351 , residues 32 to 34 of SEQ ID NO: 353, residues 24 to 26 of SEQ ID NO: 355 and residues 26 to 27 of SEQ ID NO: 763.

For example, a peptide ligand may be located in the VWV scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residues 22, 23, or 24 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 28, 29 or 30 of SEQ ID NO: 351 , residues 31 , 32 or 33 of SEQ ID NO: 353, residues 23, 24 or 25 of SEQ ID NO: 355 and residues 25 and 26 of SEQ ID NO: 763. The peptide ligand may be inserted into the VWV scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residues 24, 25, or 26 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 30, 31 or 32 of SEQ ID NO: 351 , residues 33, 34 or 35 of SEQ ID NO: 353, and residues 25, 26 or 27 of SEQ ID NO: 355 and residues 27 and 28 of SEQ ID NO: 763. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the second loop. For example, a peptide ligand may replace the loop residues corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355 and residues 26 and 27 of SEQ ID NO: 763.

In some embodiments, a grafted VWV scaffold may comprise a first peptide ligand within the first loop and a second peptide ligand within the second loop.

In some embodiments, a grafted VWV scaffold contains a first peptide ligand and a second peptide ligand in respective positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted V V scaffold contains first and second peptide ligands that do not interfere steri cally with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e. , a target protein and an E3 ligase.

Figure 39 B shows an example of a grafted VWV scaffold created by a grafting first and second peptide ligands into the first and second loops.

In some embodiments, a VWV scaffold may further comprise an alpha helix stacked against the beta-sheets of the VWV domain (termed herein a helixWW scaffold). The term“VWV scaffold” as used herein encompasses VWV scaffolds and helixWW scaffolds, unless context dictates otherwise.

A helixWW scaffold, as used herein, has a length in the amino acid range of 30 to 80 amino acids, for example 30 to 60 amino acids or 30 to 50 amino acids, and any amino acid length there between.

A helixWW scaffold may be in the MW range of 4700 kDa to 8900 kDa, typically about 5800 kDa in MW.

Suitable helixWW scaffolds useful according to the invention include the VWV domains selected from the following, without limitation: Pre-mRNA-processing protein PRP40

(helixWW domain 1 to 75; UniProtKB - P33203 (PRP40_YEAST); PDB: 106W), Peptidyl- prolyl cis-trans isomerase (helixWW domain 7 to 57; UniProtKB - Q5AZY5

(Q5AZY5_EMENI); PDB: 2JV4), Membrane-associated guanylate kinase, VWV and PDZ domain-containing protein 1 (helixWW domain 355 to 401 ; UniProtKB - Q96QZ7

(MAGI1JHUMAN); RSCB: 2YSE), Transcriptional coactivator YAP1 (helixWW domain 165 to 209; UniProtKB - P46937 (YAP1 JHUMAN); RSCB: 4REX), E3 ubiquitin-protein ligase NEDD4-like (helixWW domain 496 to 535; UniProtKB - Q96PU5 (NED4LJHUMAN); RSCB: 2LAJ). Other suitable helixWW sequences are shown in Table 37.

In some embodiments, a helixWW scaffold may comprise the amino acid sequence of SEQ ID NO: 359 or a variant thereof.

In some embodiments, the helixWW scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 360 or a variant thereof.

The a-helix of a helixWW scaffold is located within the positions corresponding to residues 30 to 43 of SEQ ID NO: 359.

The first loop of a helixWW scaffold is located within the positions corresponding to residues 51 to 53 of SEQ ID NO: 359.

The second loop of a helixWW scaffold is located within the positions corresponding to residues 61 to 63 of SEQ ID NO: 359

A grafted helixWW scaffold may contain a peptide ligand in one or more of; the first and second loop portions of the helixWW scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop) and the a-helical region of the helixWW scaffold.

A grafted helixWW scaffold may contain a peptide ligand inserted within the first loop (a “loop peptide”) for example between residues corresponding to residues 51 to 53 of SEQ ID NO: 359. For example, a peptide ligand may be located in the helixWW scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 50, 51 , or 52 of SEQ ID NO: 359. The peptide ligand may be inserted into the helixWW scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 52, 53, or 54 of SEQ ID NO: 359.

A grafted helixWW scaffold may contain a peptide ligand inserted within the first loop (a “loop peptide”) for example between residues corresponding to residues 61 to 63 of SEQ ID NO: 359. For example, a peptide ligand may be located in the helixWW scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 60, 61 or 62 of SEQ ID NO: 359. The peptide ligand may be inserted into the helixWW scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 62, 63, or 64 of SEQ ID NO: 359.

A grafted helixWW scaffold may contain a peptide ligand in the helix portion of the helixWW scaffold. A helical peptide ligand may be inserted in a helical portion of the VWV scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). A“helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold. In some embodiments, a helical peptide ligand replaces the helix of the helixWW scaffold. For example, a peptide ligand may replace residues K30, E31 , S34, N35 L38, R41 , E42 of SEQ ID NO: 359.

Suitable peptide ligands include helical peptide ligands as described herein.

In some embodiments, a grafted helixWW scaffold is created by isomorphic replacement of those residues (for example in SEQ ID NO: 359 one or more of K30, E31 , S34, N35 L38, R41 , E42) of the helix portion of the WW scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.

For example, a helix peptide ligand may comprise the amino acid sequence of SEQ ID NO: 361 , a fragment of SEQ ID NO: 361 or a variant of either of these.

Z ₁ Z ₂ LIZ ₃ Z ₄ EEZ ₅ LLZ ₆ Z ₇ N (SEQ ID NO: 361 , _where Zi to Z ₇ are independently any amino acid any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.)

In some embodiments, a grafted helixWW scaffold may contain a helical peptide ligand in the helix portion of the helixWW scaffold between positions corresponding to 30 and 43 of SEQ ID NO: 359 and a loop peptide ligand in the first loop of the helixWW scaffold between the positions corresponding to residues 51 and 53 of SEQ ID NO: 359.

In some embodiments, a grafted helixWW scaffold may contain a helical peptide ligand in the helix portion of the helixWW scaffold between positions corresponding to 30 and 43 of SEQ ID NO: 359 and a loop peptide ligand in the second loop portion of the helixWW scaffold between the positions corresponding to residues 60 and 62 of SEQ ID NO: 359.

For example, a grafted helixWW scaffold may comprise the amino acid sequence of SEQ ID NO: 362 or a variant thereof;

(SEQ NO: 362; where [Xi, X2...X _n ] and is a peptide ligand of n amino acids, where n is 3 to 30, and Xi to X _n are independently any amino acid; where Zi to Z7 are amino acids isomorphically replaced on the solvent-facing side of the helix and Zi to Zz are independently any amino acid).

Examples of grafted VWV scaffolds according to the eighth aspect of the invention are shown in Figure 42 B and Table 38.

For example, a grafted VWV scaffold may comprise a VWV scaffold with the amino acid sequence of residues 1 to 44 of SEQ ID NO: 763 (PPX124 of Table 38 without the HA Tag).

A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 16 to 17 of SEQ ID NO: 763) and the second loop (loop 2; residues 26 to 27 of SEQ ID NO: 763) of the VWV scaffold.

Preferably, the peptide ligand is located in the first loop. Preferably, the E3 ligase-binding peptide ligand is located in the second loop. Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from APC (Adenomatous polyposis coli), such as SEELEALEALELDE, SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof; and KRAS binding ligands, such as peptides from RBP, such as

SHYPWFKARLYPLS, GHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof, and peptides from KBL, such as PLYISY and PLYISYPV and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG and GLDPETGELL and variants thereof.

In some embodiments, a grafted VWV scaffold of the eighth aspect may comprise an amino acid sequence shown in Table 38 or a variant of an amino acid sequence shown in Table 38. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 38 are replaced by a different peptide ligand.

Suitable peptide ligands are described below. Variants may also include variants in which the VWV scaffold sequence in a reference amino acid sequence of Table 38 is replaced by a different VWV scaffold sequence. Suitable VWV scaffold sequences are described above.

The VWV scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the VWV scaffolds.

(ix) Fibritin Scaffolds

A ninth aspect relates to chimeric proteins that comprise Fibritin scaffolds (i.e. grafted Fibritin scaffolds). One or more peptide ligands are located in the Fibritin scaffold of the chimeric protein, for example in the coiled-coil subdomain and/or the disordered region.

The Fibritin domain is well known and well-characterized in the prior art and contains the Foldon subdomain that is responsible for the formation of the trimeric structure of Fibritin. Foldon domains have been widely studied in the area of protein folding and design. (Meier et al 2004 J Mol Biol. 344(4):1051-69; Krammer et al 2012 PLoS ONE 7(8): e43603).

The structure of Fibritin domains has been described in the art (see for example Pfam PF07921 , PDB 1 RFO, 1 U0P, 1 NAY, 4NCV, 4NCU; Guthe et al J 2004 Mol Biol 337:905- 915; Papanikolopoulou et al 2004 JBC 2004 279:8991-8998.)

The invention adopts the well understood sequence-structure relationships of the Fibritin domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a Fibritin scaffold is a folded trimer-forming protein comprising a coiled-coil sub-domain and a beta-sheet foldon subdomain linked by a disordered region.

A Fibritin scaffold, as used herein, has a length in the amino acid range of 27 to 100 amino acids, and any amino acid length there between, for example, 27 to 42 amino acids, 27 to 65 amino acids, such as 42, 65 or 90 amino acids.

A Fibritin scaffold may be in the MW range of 3-10 kDa as a monomer and 9- 30 kDa as a trimers, typically about 21.7 kDa,

A suitable Fibritin scaffold may comprise the consensus sequence;

qn+l+ssIQDlQvEIGNNnSGLKG+VIkLskdvyGTNPnGdTVEErGIkkTVKdlyTalg aGklpdAPs

DieGkaYVRekDGAwvkL+ti (SEQ ID NO: 410; wherein upper case indicates high conservation; lower case indicates low conservation; + indicates that not all sequences have a residue at that position)

Suitable Fibritin scaffolds useful according to the invention may include the Fibritin domain of bacteriophage T4 Fibritin (ADJ39878.1 ; Gene ID: 1258630, NP_049771.1 ; residues 426- 484) or variants thereof. Other Fibritin scaffolds useful according to the invention may include the sequences shown in Table 39 and Table 40 (SEQ ID NOs 371-409) and variants thereof.

The following PDB codes from the Research Col laboratory for Structural Bioinformatics (RCSB) protein data bank (https://www.rcsb.org/; Berman et al P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242) identify representative structures of suitable Fibritin scaffolds useful according to the invention without any limitation: 1 RFO, 2WW7, 1 U0P, 2VWV6, 4NCV, 4NCW, 1V1 H, 1V1 I, 2J5A, 10X3, and 4NCU. Other suitable Fibritin scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids

Research (2016) Database Issue 44:D279-D285).

In some embodiments, a Fibritin scaffold may comprise the amino acid sequence of SEQ ID NO: 363 or a variant thereof.

The Fibritin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 364 or a variant thereof.

In some embodiments, a Fibritin scaffold may comprise the amino acid sequence of SEQ ID NO: 365 or a variant thereof.

(SEQ ID NO 365; b sheet solid

underline; a helix dotted underline)

The Fibritin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 366 or a variant thereof.

Preferred Fibritin scaffolds lack lysine residues, for example to avoid ubiquitination. For example, the lysine residues in a Fibritin domain may be replaced by E, Q or R to generate a Fibritin scaffold. The Fibritin domain of SEQ ID NO: 363 has three Lys residues (Lys16, Lys22 and Lys33). In some embodiments, Lys 16 of the Fibritin domain may be replaced with arginine. In some embodiments, the residues Lys22 of the Fibritin domain may be replaced with arginine. In some embodiments, the residues Lys33 of the Fibritin domain may be replaced with arginine. A suitable lysine-free Fibritin scaffold may comprise the amino acid sequence of SEQ ID NO: 367 or a variant thereof.

A lysine- free Fibritin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 368 or a variant thereof.

The coiled-coil subdomain of a Fibritin scaffold is located within the positions corresponding to residues 1 to 38 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 15 of SEQ ID NO: 365.

The disordered region of a Fibritin scaffold is located within the positions corresponding to residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 16 to 27 of SEQ ID NO: 365.

The b-sheet (foldon) subdomain of a Fibritin scaffold is located within the positions corresponding to residues 51 to 60 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 28 to 37 of SEQ ID NO: 365.

Peptide ligands may be inserted into one or both of the coiled-coil subdomain and the disordered region in the Fibritin scaffold. Preferably, peptide ligands are not inserted into the b-sheet (foldon) subdomain.

A grafted Fibritin scaffold contains a peptide ligand in one or both of the coiled-coil subdomain and the disordered region of the Fibritin scaffold. The peptide ligands may be heterologous.

In some embodiments, a grafted Fibritin scaffold may contain a peptide ligand within the disordered region that corresponds to residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 16 to 27 of SEQ ID NO: 365. For example, a loop peptide ligand may be located in the Fibritin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 38, 39, 40,

41 , 42, 43, 44, 45, 46, 47, 48 or 49 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 15,

16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 26 of SEQ ID NO: 365. The peptide ligand may be inserted into the Fibritin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 40, 41 , 42, 43,44, 45,46, 47, 48, 49, 50 or 51 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues

17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO: 365.

Peptide ligands may be inserted into one or both of the coiled-coil subdomain and the disordered region in the Fibritin scaffold. Preferably, peptide ligands are not inserted into the b-sheet (foldon) subdomain.

In other embodiments, a grafted Fibritin scaffold contains a peptide ligand in one or more of a first helix, a first loop and a second loop of the Fibritin scaffold. The peptide ligands may be heterologous.

A grafted Fibritin scaffold may contain a peptide ligand within the first helix that corresponds to residues 7 to 14 of SEQ ID NO: 772, the first loop that corresponds to residues 39 to 40 of SEQ ID NO: 772 or the second loop that corresponds to residues 96 to 97 of SEQ ID NO: 772. For example, a loop peptide ligand may be located in the Fibritin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 6, 7, 8, 9, 10, 11 , 12 or 13; residue corresponding to 38 or 39; or the residue corresponding to residue 95 or 96 of SEQ ID NO: 772. The peptide ligand may be inserted into the Fibritin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 15, 14, 13, 12, 11 , 10, 9 or 8 of SEQ ID NO: 772; residues 41 or 40 of SEQ ID NO: 772 or residues 98 or 97 of SEQ ID NO: 772.

Preferably, a grafted Fibritin scaffold may contain a peptide ligand within the disordered region that corresponds to residues 48 to 49 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 25 to 26 of SEQ ID NO: 365. The loop peptide ligand may be located in the Fibritin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 47 or 48 of SEQ ID NO: 363 or SEQ ID NO: 367 or residue 24 or 25 of SEQ ID NO: 365. The peptide ligand may be inserted into the Fibritin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 49 or 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residue 26 or 27 of SEQ ID NO: 365. The peptide ligand may be added to the disordered region or may replace one or more residues of the disordered region. For example, the peptide ligand may replace the disordered region.

A modelled structure of the longer (44A; SEQ ID NO: 363) and shorter (44 B; SEQ ID NO: 365) Fibritin domains with a grafted loop is shown in Figure 44.

In some embodiments, a grafted Fibritin scaffold may contain a helical peptide ligand within the coiled-coil subdomain. A helical peptide ligand may be inserted in a helical portion of the Fibritin scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure), such as residues 7 to 14 of SEQ ID NO: 772. A “helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold. In some embodiments, a helical peptide ligand replaces the helix of the Fibritin scaffold. For example, a peptide ligand may replace one or more of residues 1 to 38 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 15 of SEQ ID NO: 365 or residues 7 to 14 of SEQ ID NO: 772. In some embodiments, a grafted Fibritin scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to any one of residues 1 to 37 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 14 of SEQ ID NO: 365 or residues 6 to 13 of SEQ ID NO: 772. In some embodiments, a grafted Fibritin scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to any one of residues 2 to 39 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 2 to 16 of SEQ ID NO: 365 or residues 8 to 15 of SEQ ID NO: 772. The peptide ligand may be added to the coiled-coil subdomain or may replace one or more residues of the coiled-coil subdomain. For example, the peptide ligand may replace the coiled-coil subdomain.

Suitable peptide ligands include helical peptide ligands as described herein. For example, a helical peptide ligand may comprise SEQ ID NO: 369 or SEQ ID NO: 370 or a fragment thereof, for example 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous residues of the amino acid sequence of SEQ ID NO: 369 or SEQ ID NO: 370.

369; where Xi to X ₂₄ are each independently any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine. (SEQ ID NO: 370; where Xi to X _g are each independently any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.

In some embodiments, a grafted Fibritin scaffold is created by isomorphic replacement of those residues (‘X above) of the helix portion of the Fibritin scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent-facing residues of the helix peptide ligand.

In some preferred embodiments, a grafted Fibritin scaffold may contain a helical peptide ligand in the coiled-coil subdomain of the Fibritin scaffold between positions corresponding to 1 and 38 of SEQ ID NOs: 363 and 367 and a loop peptide ligand in the disordered region of the Foldon scaffold for example between the positions corresponding to residues 48 and 49 of SEQ ID NOs: 363 and 367 or residues 25 and 26 of SEQ ID NO: 365.

Examples of grafted Fibritin scaffolds according to the ninth aspect of the invention are shown in Figure 47B and Table 41.

For example, a grafted Fibritin scaffold may comprise a Fibritin scaffold with the amino acid sequence of residues 1 to 121 of SEQ ID NO: 772 (PPX133 of Table 41 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the coiled-coil subdomain and the disordered subdomain of the Fibritin scaffold.

Preferably, the peptide ligand is located in the first helix, the first loop or the second loop. Preferably, the E3 ligase-binding peptide ligand is located in the first loop or the second loop.

For example, the target-binding peptide ligand may be in the first helix and the E3 ligase- binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop; or the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop.

Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

and variants thereof; and KRAS binding ligands, for example peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof and sequences from the protein SOS1 (Son of sevenless homolog 1), such as IAETNFRKYAE, LTNxLK, TNVLKLQE, TNxxKxxE or IxxTNxxKTxE and variants thereof, and peptides from RBP, such as

SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and HYPWFKARLYPL and variants thereof.

Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG and GLDPETGELL and variants thereof.

In some embodiments, a grafted Fibritin scaffold of the ninth aspect may comprise an amino acid sequence shown in Table 41 or a variant of an amino acid sequence shown in Table 41. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 41 are replaced by a different peptide ligand.

Suitable peptide ligands are described below. Variants may also include variants in which the Fibritin scaffold sequence in a reference amino acid sequence of Table 41 is replaced by a different Fibritin scaffold sequence. Suitable Fibritin scaffold sequences are described above.

The Fibritin scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Fibritin scaffolds.

(x) aPP Scaffolds

A tenth aspect relates to chimeric proteins that comprise avian pancreatic polypeptide (aPP) scaffolds (i.e. grafted aPP scaffolds). One or more peptide ligands are located in the aPP scaffold of the chimeric protein, for example at one or both of the loop and the helical region. Avian pancreatic polypeptide (aPP) scaffold is a small endocrine protein that has been previously used in protein engineering (Chin et al (2001) Bioorg Med Chem Lett 11 (12) 1501-1505; McGee et al (2018) JBC 293(9) 3265-3280; Hodges et al (2007) J Am Chen Soc 129 1 1024-11025). The aPP scaffold is sufficiently small to be synthetically tractable (hence allowing incorporation of unnatural amino acids) as well as genetically encodable.

The structure of the aPP domain is well known in the art (see for example PFAM 00159;

PDB 1 PPT; Blundell et al (1981) PNAS 78: 4175-4179; Hodges et al J. Am. Chem. Soc., 2007, 129 (36), pp 1 1024-1 1025; Applebaum et al (2012) Chem Biol. 2012 Jul 27; 19(7): 819-830).

The invention adopts the well understood sequence-structure relationships of aPP domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, an aPP scaffold is a folded protein comprising a left-handed poly-proline-ll- like helix, linked by a beta-turn to an alpha-helix (i.e. an aPP domain structure).

An aPP scaffold, as used herein, has a length in the amino acid range of 25-55 amino acids, for example 25-45 amino acids, 30 to 40 amino acids, or 25 to 36 amino acids, and any amino acid length there between, for example about 32 amino acids.

An aPP scaffold may be 2750-5370 kDa, for example 2750-5340 kDa or 2750-4240 kDa and may be typically about 4239 kDa in MW,

Suitable aPP scaffolds useful according to the invention include the aPP domains shown in Tables 42 to 44, or variants of any one of these. A suitable aPP scaffold may for example comprise the consensus sequence of SEQ ID NO: 41 1. where upper case

indicates high conservation; lower case indicates low conservation; + indicates that not all sequences have a residue at that position).

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable aPP scaffolds useful according to the invention without any limitation: 2H4B, 2H3S, 1 PPT and 2BF9. Suitable aPP scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids

Research (2016) Database Issue 44:D279-D285). Other suitable aPP scaffolds include residues 1 to 47 of SEQ ID NO: 784 and residues 1 to 43 of SEQ ID NO: 788.

In some embodiments, an aPP scaffold may comprise the amino acid sequence of SEQ ID NO: 412 or a variant thereof. polyproline II helix dotted underlined, a helix double underlined)

The aPP scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 413 or a variant thereof.

Preferred aPP scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in an aPP domain may be replaced by E, N, Q , S or R to generate an aPP scaffold. A suitable lysine-free aPP scaffold may comprise the amino acid sequence of SEQ ID NO: 412 or a variant thereof.

In some embodiments, an aPP scaffold may display binding activity. For example an aPP scaffold may bind to a target molecule, such as KRAS, in the absence of an inserted peptide ligand. Residues other than Pro 2, Pro5, Pro8, Leu 17, Phe/Tyr20, Leu24 and Tyr 27 may be altered to modulate the binding activity of the aPP scaffold. For example, an aPP scaffold may comprise the amino acid sequence of SEQ ID NO: 414.

x ₂₉ (SEQ ID NO: 414; ; loop solid underlined; where Xi to X _2g are independently any amino acid, for example, independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine, phospho- serine, phospho-threonine and phospho-tyrosine, acetylated amino acids)

An aPP scaffold that binds to KRAS may comprise the amino acid sequence of SEQ ID NO: 415 or a variant thereof.

GPRRPRX _{IPGDDASIEDLHEYWARLWNYLYAVA} (SEQ ID NO: 415, where X1 is Cys or Sec, preferably Cys; ; loop solid underlined, polyproline II helix dotted underlined, a helix double underlined)

The aPP scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 416 or a variant thereof.

Suitable methods of generating aPP scaffolds that bind to target molecules are established in the art (Kritzer et al (2006) ChemBioChem 7 29-31 ; McGee et al (2018) JBC 293(9) 3265- 3280).

The loop of an aPP scaffold is located within the positions corresponding to residues 9 to 13 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415; residues 11 to 12 of SEQ ID NO: 784 and residues 13 to 14 of SEQ ID NO: 788.

The helix of an aPP scaffold is located within the positions corresponding to residues 14 to 32 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415; residues 21 to 32 of SEQ ID NO: 784 and residues 23 to 30 of SEQ ID NO: 788.

A grafted aPP scaffold may comprise a peptide ligand in the loop portion of the aPP scaffold corresponding to residues 9 to 13 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415; residues 11 to 12 of SEQ ID NO: 784 or residues 13 to 14 of SEQ ID NO: 788, for example, inserted into a specific location of a loop (thus providing an extended loop). The peptide ligand may be heterologous.

For example, a peptide ligand may be located in the aPP scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 8, 9, 10, 11 , or 12 of SEQ ID NOs: 412, 414 or 415, preferably the residue corresponding to residue 8, 9, 10 or 11 of SEQ ID NOs: 412, 414 or 415 or the residue corresponding to residue 10 or 11 of SEQ ID NO: 784 or residue 12 or 13 of SEQ ID NO: 788. The peptide ligand may be inserted into the aPP scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 10, 11 , 12, 13, or 14 of SEQ ID NOs: 412, 414 or 415, preferably the residue corresponding to residue 10, 11 , 12, or 13 of SEQ ID NOs: 412, 414 or 415 or residue 12 or 13 of SEQ ID NO: 784 and residue 14 or 15 of SEQ ID NO: 788. The peptide ligand may be added to the loop or may replace one or more residues of the loop. For example, the peptide ligand may replace the loop. For example, a peptide ligand may replace the loop residues corresponding to residues 9 to 13 of SEQ ID NOs: 412, 414 or 415; residues 11 to 12 of SEQ ID NO: 784 and residues 13 to 14 of SEQ ID NO: 788.

In some embodiments, a grafted aPP scaffold may comprise an aPP scaffold that binds to a target molecule and a peptide ligand located within the loop located at positions corresponding to residues 9 to 13 of SEQ ID NOs: 412, 414 or 415. The modelled structure of an aPP scaffold with a grafted loop is shown in Figure 49.

In other embodiments, a grafted aPP scaffold may comprise an aPP scaffold comprising a first peptide ligand located within the loop located at positions corresponding to residues 9 to 13 of SEQ ID NOs: 412, 414 or 415 or residues 11 to 12 of SEQ ID NO: 784 or residues 13 to 14 of SEQ ID NO: 788 and a second peptide ligand located within the helical region located at positions corresponding to residues 14 to 32 of SEQ ID NOs: 412, 414 or 415 or residues 21 to 32 of SEQ ID NO: 784 and residues 23 to 30 of SEQ ID NO: 788.

A grafted aPP scaffold may comprise a peptide ligand in the helical region of the aPP scaffold. A helical peptide ligand may be inserted in a helical portion of the aPP scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). A“helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold. In some embodiments, a helical peptide ligand replaces the helix of the aPP scaffold. For example, a peptide ligand may replace residues 14 to 32 of SEQ ID NOs: 412, 414 or 415 or residues 21 to 32 of SEQ ID NO: 784 or residues 23 to 30 of SEQ ID NO: 788. For example, a peptide ligand may replace one or more of residues V14, E15, D16, 118, R19, N21 , D22, Q24, Q25, L27, N29, V29, V30 and T31 of SEQ ID NO: 412. In some embodiments, a grafted aPP scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 of SEQ ID NOs: 412, 414 or 415. In some embodiments, a grafted aPP scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 ,

32, or 33 of SEQ ID NOs: 412, 414 or 415.

Suitable peptide ligands include helical peptide ligands as described herein.

In some embodiments, a grafted aPP scaffold is created by isomorphic replacement of those residues (for example in SEQ ID NO: 412 one or more of V14, E15, D16, 118, R19, N21 ,

D22, Q24, Q25, L27, N29, V29, V30 and T31) of the helix portion of the aPP scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.

For example, a helix peptide ligand may comprise the amino acid sequence of SEQ ID NO: 417, a fragment of SEQ ID NO: 417 or a variant of either of these. (SEQ ID NO: 417, where Xi to X are

independently any amino acid any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.)

In other embodiments, a chimeric protein may comprise;

(i) a first grafted aPP scaffold having a first peptide ligand located within the loop of the first aPP scaffold located at a position corresponding to residues 9 to 13 of SEQ ID NOs: 412, 414 or 415 and

(ii) a second aPP scaffold having a second peptide ligand located within the loop of the second aPP scaffold located at a position corresponding to residues 9 to 13 if SEQ ID NOs: 412, 414 or 415.

In other embodiments, a chimeric protein may comprise;

(i) a first grafted aPP scaffold having a first peptide ligand located within the loop of the first aPP scaffold located at a position corresponding to residues 9 to 13 of SEQ ID NOs: 412, 414 or 415, residues 11 to 12 of SEQ ID NO: 784 or residues 13 to 14 of SEQ ID NO: 788and

(ii) a second aPP scaffold having a second peptide ligand located within the helical region of the second aPP scaffold located at a position corresponding to residues 14 to 32 of SEQ ID NOs: 412, 414 or 415; residues 21 to 32 of SEQ ID NO: 784 or residues 23 to 30 of SEQ ID NO: 788.

Generally in aPP scaffolds, the peptide ligands are preferably 20 angstroms apart, they may be 19 angstroms, 18 angstroms, 17 angstroms, 16 angstroms but no less than 15 angstroms apart. A person of skill in art can use 3D software such as Chimera or Pymol to determine the minimum distances between positions for ideal positioning in three dimensional orientation

The first and the second peptide ligands may bind to the same target molecule or more preferably to different target molecules.

In some embodiments, the first and the second aPP scaffolds may be comprised in a single amino acid chain. For example, the first and the second aPP scaffolds may be connected through a peptidyl bond, either directly or through a linker sequence, Suitable linker sequences are well known in the art and include short flexible peptide linkers, such as (GS) _n , where n is 2 or 4.

In other embodiments, a chimeric protein may comprise first and the second aPP scaffolds that are connected through a disulfide linkage.

The aPP scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the aPP scaffolds.

In other embodiments, the aPP scaffold of the chimeric protein may also display binding activity i.e. the aPP scaffold may mediate binding of the chimeric protein to a first target molecule. The peptide ligand may mediate binding of the chimeric protein to a second target molecule.

Examples of grafted aPP scaffolds according to the tenth aspect of the invention are shown in Figure 51 and Table 45 (SEQ ID NOs 777-798).

For example, a grafted aPP scaffold may comprise an aPP scaffold with the amino acid sequence of residues 1 to 47 of SEQ ID NO: 784 (PPX139 of Table 45 without the HA Tag)

In some embodiments, a grafted aPP scaffold may comprise E3 ligase-binding peptide ligand, for example a MDM2 binding sequence from p53, such as FSxxWxxL, FxxXWxxL, and variants thereof. The E3 ligase-binding peptide ligand may be endogenous to the aPP scaffold. For example, an E3 ligase-binding peptide ligand may be located at residues 23 to 30 of SEQ ID NO: 784. A target-binding peptide ligand may be grafted into the first loop of the aPP scaffold (loop 1 residues 11 to 12 of SEQ ID NO: 784).

In other embodiments, a grafted aPP scaffold may comprise an aPP scaffold with the amino acid sequence of residues 1 to 43 of SEQ ID NO: 788 (PX163 of Table 45 without the HA Tag). A target-binding peptide ligand and E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 13 to 14 of SEQ ID NO: 788) and the helix (helix 1 ;

residues 23 to 30 of SEQ ID NO: 788) of the aPP scaffold. Preferably, the peptide ligand is located in the helix. Preferably, the E3 ligase-binding peptide ligand is located in the first loop. For example, the target-binding peptide ligand may be in the helix and the E3 ligase- binding peptide ligand may be in the first loop. Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof; peptides from the protein AXIN, such as ILxxHV, AxxILDxHV or ILDxHV and variants thereof, peptides from BCL9, such as TLxxlQxxL, LxTLxxlQ, and SLxxlxxML and variants thereof, and KRAS binding ligands, for example peptides from KBL, such as PLYISY, PLYISYDPV and

PLYISYPV and variants thereof, peptises from the protein SOS1 (Son of sevenless homolog 1), such as IAETNFRKYAE, LTNxLK, TNVLKLQE, TNxxKxxE or IxxTNxxKTxE and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and HYPWFKARLYPL and variants thereof.

Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG and GLDPETGELL and variants thereof, binding sequences from p27, such as AGSNEQEPNR and variants thereof, and peptides from Wnk4, such as EEPEADQH and variants thereof.

In some embodiments, a grafted aPP scaffold of the tenth aspect may comprise an amino acid sequence shown in Table 45 or a variant of an amino acid sequence shown in Table 45. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 45 are replaced by a different peptide ligand.

Suitable peptide ligands are described below. Variants may also include variants in which the aPP scaffold sequence in a reference amino acid sequence of Table 45 (SEQ ID NOs 777-798) is replaced by a different aPP scaffold sequence. Suitable aPP scaffold sequences are described above.

(xi) Fibronectin Scaffolds

An eleventh aspect relates to chimeric proteins that comprise fibronectin (FN3) scaffolds (i.e. grafted fibronectin scaffolds). One or more peptide ligands are located in the fibronectin scaffold of the chimeric protein, for example in one or more of the first, second, third and fourth loops.

The type III domain of fibronectin (FN3) is a conserved domain that is found in numerous proteins across many species.

The structure of the Fibronectin type III domain is well-known in the art (see for example, Pfam PF00041 ; PDB 3UTO; SM00060; Bazan JF, PNAS USA 1990; 87:6934-6938) The invention adopts the well understood sequence/structure relationships of the fibronectin type III domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a fibronectin scaffold is a protein comprising a b sandwich fold with one b sheet containing four strands and the other sheet containing three strands.

A fibronectin scaffold as used herein may have a length in the amino acid range of 80 to 110 amino acids, for example, 80-100 amino acids, 85-95 amino acids, or 95-110 amino acids, and any amino acid length there between, for example about 90 amino acids.

A FN3 scaffold may have a MW of 8800-11000 Da, for example 9450- 10600 Da, typically about 10050 Da.

Suitable FN3 scaffolds useful according to the invention include FN3 domains selected from the following human proteins, without limitation: ABI3BP; ANKFN1 ; ASTN2; AXL; BOC; BZRAP1 ; C20orf75; CDON; CHL1 ; CMYA5; CNTFR; CNTN1 ; CNTN2; CNTN3; CNTN4; CNTN5; CNTN6; COL12A1 ; COL14A1 ; COL20A1 ; COL7A1 ; CRLF1 ; CRLF3; CSF2RB; CSF3R; DCC; DSCAM; DSCAML1 ; EBI3; EGFLAM; EPHA1 ; EPHA10; EPHA2; EPHA3; EPHA4; EPHA5; EPHA6; EPHA7; EPHA8; EPHB1 ; EPHB2; EPHB3; EPHB4; EPHB6;

EPOR; FANK1 ; FLRT1 ; FLRT2; FLRT3; FN1 ; FNDC1 ; FNDC3A; FNDC3B; FNDC4; FNDC5; FNDC7; FNDC8; FSD1 ; FSD1 L; FSD2; GHR; HCFC1 ; HCFC2; HUGO; IFNGR2; IGF1 R; IGSF22; IGSF9; IGSF9B; IL4R; IL11 RA; IL12B; IL12RB1 ; IL12RB2; IL20RB; IL23R; IL27RA; IL31 RA; IL6R; IL6ST; IL7R; INSR; INSRR; ITGB4; II6ST; KAL1 ; KALRN; L1CAM; LEPR; LIFR; LRFN2; LRFN3; LRFN4; LRFN5; LRIT1 ; LRRN1 ; LRRN3; MERTK; MIDI ; MID2; MPL; MYBPC1 ; MYBPC2; MYBPC3; MYBPH; MYBPHL; MYLK; MYOM1 ; MYOM2; MYOM3; NCAM1 ; NCAM2; NE01 ; NFASC; NOPE; NPHS1 ; NRCAM; OBSCN; OBSL1 ; OSMR;

PHYHIP; PHYHIPL; PRLR; PRODH2; PTPRB; PTPRC; PTPRD; PTPRF; PTPRG; PTPRH; PTPRJ; PTPRK; PTPRM; PTPRO; PTPRS; PTPRT; PTPRU; PTPRZ1 ; PTPsigma; PUNC; RIMBP2; ROB01 ; ROB02; ROB03; ROB04; ROS1 ; SDK1 ; SDK2; SNED1 ; SORL1 ; SPEG; TEK; TIE1 ; TNG; TNN; TNR; TNXB; TRIM36; TRIM42; TRIM46; TRIM67; TRIM9; TTN; TYR03; UMODL1 ; USH2A; VASN; VWA1 ; dJ34F7.1 ; fmi, or variants of any one of these.

Other suitable FN3 domains include residues 802-891 of UniProtKB P24821-2, P24821-3, P24821-4, P24821-5, P24821-6, P24821 , Q29116-3, Q29116-2, Q29116, Q80YX1-2, Q80YX1-3, Q80YX1-4, Q80YX1-5 and Q80YX1 ; and residues 773-862 of UniProtKB P10039-3, P 10039-2 and P10039 (see Table 47).

Examples of suitable FN3 scaffolds are shown in Table 46.

The following PDB codes from the Research Col laboratory for Structural Bioinformatics (RCSB) protein data bank (https://www.rcsb.org/; Berman et al P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242) identify representative structures of suitable FN3 scaffolds useful according to the invention without any limitation: 5KF4, 3WIH, 2DM4, 5J7C, 2EE2, 2DMK, 2DB8, 1WFN, 1WFO, 1WJ3, 2VKW, 5E4Q, and 2EDY.

Suitable FN3 scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44:D279-D285).

Suitable FN3 scaffolds also include residues 1 to 96 of SEQ ID NO: 801.

In some embodiments, a fibronectin scaffold may comprise the amino acid sequence of SEQ ID NO: 418 or a variant thereof.

The fibronectin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 419 or a variant thereof.

Preferred fibronectin scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in a fibronectin domain may be replaced by S, R, P, or E to generate a Lys-free fibronectin scaffold. The fibronectin domain of SEQ ID NO: 418 has three Lys residues (Lys11 , Lys24, Lys39, Lys 63 and Lys85). In some embodiments, Lys 11 may be replaced by S or R. In some embodiments, Lys24 may be replaced by P or R. In some embodiments, Lys39 may be replaced by E or R. In some embodiments, Lys 63 may be replaced by E or R. In some embodiments, Lys85 may be replaced by R. A suitable Lys-free fibronectin scaffold may comprise the amino acid sequence of SEQ ID NO: 420 or a variant thereof.

The Lys-free fibronectin scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 421 or a variant thereof.

The first loop of a fibronectin scaffold is located within the positions corresponding to residues 14 to 15 of SEQ ID NO: 418 or SEQ ID NO: 420.

The second loop of a fibronectin scaffold is located within the positions corresponding to residues 25 to 26 of SEQ ID NO: 418 or SEQ ID NO: 420.

The third loop of a fibronectin scaffold is located within the positions corresponding to residues 43 to 44 of SEQ ID NO: 418 or SEQ ID NO: 420 or residues 41 to 42 of SEQ ID NO: 8.

The fourth loop of a fibronectin scaffold is located within the positions corresponding to residues 81 to 82 of SEQ ID NO: 418 or SEQ ID NO: 420 or residues 41 to 42 of SEQ ID NO: 8.

Peptide ligands may be located in one or two or more of the first, second, third and fourth loops in the fibronectin scaffold. Preferably, peptide ligands are located in the second and third loops in the fibronectin scaffold.

A grafted fibronectin scaffold contains a peptide ligand in a loop portion of the fibronectin scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). The peptide ligand may be heterologous.

In some embodiments, a grafted fibronectin scaffold may contain a peptide ligand within the first loop (a“loop peptide”). For example, a peptide ligand may be located in the fibronectin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 13 or 14 of SEQ ID NO: 418 or SEQ ID NO: 420. The peptide ligand may be inserted into the fibronectin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 15 or 16 of SEQ ID NO: 418 or SEQ ID NO: 420. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 14 to 15 of SEQ ID NO: 418 or SEQ ID NO: 420.

In some embodiments, a grafted fibronectin scaffold may contain a peptide ligand within the second loop (a“loop peptide”). For example, a peptide ligand may be located in the fibronectin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 25 of SEQ ID NO:

418 or SEQ ID NO: 420. The peptide ligand may be inserted into the fibronectin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 26 of SEQ ID NO: 418 or SEQ ID NO: 420. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the second loop. For example, a peptide ligand may replace the loop residues corresponding to residues 25 to 26 of SEQ ID NO: 418 or SEQ ID NO: 420.

In some embodiments, a grafted fibronectin scaffold may contain a peptide ligand within the third loop (a“loop peptide”). For example, a peptide ligand may be located in the fibronectin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 42 of SEQ ID NO: 418 or SEQ ID NO: 420 or the residue corresponding to 42 or 43 of SEQ ID NO: 801. The peptide ligand may be inserted into the fibronectin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 43 of SEQ ID NO: 418 or SEQ ID NO: 420 or the residue corresponding to 40 or 41 of SEQ ID NO: 801. The peptide ligand may be added to the third loop or may replace one or more residues of the third loop. For example, the peptide ligand may replace the third loop. For example, a peptide ligand may replace the loop residues corresponding to residues 43 to 44 of SEQ ID NO: 418 or SEQ ID NO: 420 or the loop residues corresponding to residues 41 to 42 of SEQ ID NO: 801.

In some embodiments, a grafted fibronectin scaffold may contain a peptide ligand within the fourth loop (a“loop peptide”). For example, a peptide ligand may be located in the fibronectin scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 80 or 81 of SEQ ID NO: 418 or SEQ ID NO: 420 or the residue corresponding to 74, 75 or 76 of SEQ ID NO: 801. The peptide ligand may be inserted into the fibronectin scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 82 or 83 of SEQ ID NO: 418 or SEQ ID NO: 420 or the residue corresponding to 76, 77 or 78 of SEQ ID NO: 801. The peptide ligand may be added to the fourth loop or may replace one or more residues of the fourth loop. For example, the peptide ligand may replace the fourth loop. For example, a peptide ligand may replace the loop residues corresponding to residues 81 to 82 of SEQ ID NO: 418 or SEQ ID NO: 420 or the residue corresponding to residues 75 to 77 of SEQ ID NO: 801.

In some preferred embodiments, a grafted fibronectin scaffold may comprise a peptide ligand within the second and third loops.

In other preferred embodiments, a grafted fibronectin scaffold may comprise a peptide ligand within the third and fourth loops, for example at locations corresponding to residues 41 to 42 and 75 to 77 of SEQ ID NO: 801.

In some embodiments, a grafted FN3 scaffold may comprise a first peptide ligand within the second loop and a second peptide ligand within the third second loop. For example, a grafted FN3 scaffold may comprise the amino acid sequence of SEQ ID NO: 422 or a variant thereof.

RLDAPSQIEVKDVTDTTALITWFKP [XiX ₂ ...X _n ] LAEIDGIELTYGIKDVP [YiY ₂ ...Y _n ]

GDRTTiDLTEDENQYSiGNLKPDTEYEvsLiSRRGDMSSNPAKETFTT (SEQ NO: 422; where

[X ₁ X ₂ ... X _n ] and [YiY ₂ ...Y _n ] are independently peptide ligands of n amino acids, where n is 3 to 30, and Yi to Y _n and Xi to X _n are independently any amino acid, for example,

independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine, phospho-serine, phospho-threonine and phospho-tyrosine, acetylated amino acids.

The fibronectin scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the fibronectin scaffolds.

Examples of grafted fibronectin scaffolds according to the eleventh aspect of the invention are shown in Figure 53B and Table 48 (SEQ ID NOs 799-801). For example, a grafted fibronectin scaffold may comprise a fibronectin scaffold with the amino acid sequence of residues 1 to 96 of SEQ ID NO: 801 (PPX144 of Table 48 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 41 to 42 of SEQ ID NO: 801) and second loop (loop 2; residues 75 to 77 of SEQ ID NO: 801) of the fibronectin scaffold.

Preferably, the peptide ligand is located in the second loop. Preferably, the E3 ligase-binding peptide ligand is located in the first loop.

Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG, GLDPETGELLS and GLDPETGELL and variants thereof.

In some embodiments, a grafted fibronectin scaffold of the eleventh aspect may comprise an amino acid sequence shown in Table 48 or a variant of an amino acid sequence shown in Table 48. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 48 (SEQ ID NOs 799-801) are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the fibronectin scaffold sequence in a reference amino acid sequence of Table 48 is replaced by a different fibronectin scaffold sequence. Suitable fibronectin scaffold sequences are described above.

(xii) Zn Finger Scaffolds

A twelfth aspect relates to chimeric proteins that comprise Zn finger scaffolds (i.e. grafted Zn finger scaffolds). One or more peptide ligands are located in the Zn finger scaffold of the chimeric protein, for example at Zn finger and Zn finger.

A zinc (Zn) finger domain is common structural domain found in mammalian transcription factors and other proteins, and is characterised by the coordination of a Zn ion to cysteine and histidine residues within the protein. Zinc finger domains may for example comprise the sequence C-x(2,4)-C-x(3)-[LIVMFYWC]-x(8)-H-x(3,5)-H, where x is any amino acid.

The structure of the zinc finger domain is well-known in the art (see for example, Pfam PF00096; SM00355; PDOC00028; Boehm S et al Nucleic Acids Res 1997 25 2464-2469; Rosenfeld et al (1993) J. Biomol. Struct. Dyn. 11 :557-570)

The invention adopts the well understood sequence/structure relationships of zinc finger domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a zinc finger scaffold is a protein comprising a b hairpin and an a helix (bba fold) stabilized by the coordination of a Zn ion to cysteine and histidine residues (i.e. a Cys HiS Zn finger structure)) within the protein.

A zinc finger scaffold may have a length in the length range of 20-50 amino acids in length, 30-40, 35-45, or any length there between, for example about 30 amino acids.

A Zn finger scaffold may have a molecular weight (MW) in range of 2400-5800 Da, for example 3580-4690 Da or 4050-5100 Da, typically about 3590 Da.

Suitable Zn finger scaffolds useful according to the invention include Zn finger domains selected from the following human proteins, without limitation: EGR1 (zif268), basonuclin, BCL-6/LAZ-3, erythroid krueppel-like transcription factor, transcription factors Sp1 , Sp2 (3), Sp3 and Sp4, transcriptional repressor YY1 , Wilms' tumor protein, EGR1/Krox24,

EGR2/Krox20, EGR3/Pilot, EGR4/AT133, Evi-1 , GLI1 , GLI2, GLI3, HIV-EP1/ZNF40, HIV- EP2 , KR1 , KR2, KR3, KR4, KR5, HF.12, REX-1 , ZfX, ZfY, Zfp-35, ZNF7, ZNF8, ZNF35, ZNF42/MZF-1 , ZNF43, ZNF46/Kup, ZNF76, ZNF91 , and ZNF133.

Other suitable Zn finger scaffolds include residues 603-627 and 633-657 of ACE2 (P21192 UniProtKB), residues 104-126 and 132-155 of ADR1 (P07248), residues 222-244 and 306- 328 of B5RIE4 (P07247), residues 720-743 and 928-951 of BNC1 (Q01954), residues 350- 375 of BRLA (W6QV39), residues 74-97, 366-388, 402-423, 29-451 and 485-508 of CF2 (P20385).

Examples of suitable zinc finger scaffolds include residues 336 to 365 of EGR1 (zif268; NP_001955.1) or variants thereof. Other examples of suitable Zn finger scaffolds are shown in Tables 49 and 50.

The following PDB codes from the Research Col laboratory for Structural Bioinformatics (RCSB) protein data bank (https://www.rcsb.org/: Berman et al P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242) identify representative structures of suitable Zn finger scaffolds useful according to the invention without any limitation: 2YRK, 3IUF, 3MJH, 1ZR9, 2EPU, 2EPT, 2YTB, 2YTA, 5Y0U, and 2EPC.

Suitable Zn finger scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44:D279-D285).

In some embodiments, a zinc finger scaffold may comprise the amino acid sequence of SEQ ID NO: 423 or a variant thereof.

(SEQ ID NO: 423; Zn coordinating residues are

highlighted)

The zinc finger scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 424 or a variant thereof.

Preferred Zn finger scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in a zinc finger domain may be replaced by a residue other than lysine, such as S, R, P, or E, to generate a Lys-free Zn finger scaffold.

The first loop of a Zn finger scaffold is located between the Zn coordinating cysteine residues i.e. within the positions corresponding to residues 6 to 9 of SEQ ID NO: 423.

The second loop of a Zn finger scaffold is located between Zn coordinating cysteine and histidine residues i.e. within the positions corresponding to residues 11 to 12 of SEQ ID NO: 423.

The helical region of a Zn finger scaffold is located within the positions corresponding to residues 17 to 28 of SEQ ID NO: 423.

Peptide ligands may be located one or both of the first loop and the helical region; the second loop and the helical region; or the first and second loops of the Zn finger scaffold

A grafted Zn finger scaffold contains a peptide ligand in a loop portion of the Zn finger scaffold, for example, inserted into a specific location of the first or second loop (thus providing an extended loop). The peptide ligand may be heterologous.

A grafted Zn finger scaffold may contain a peptide ligand within the first loop, between the Zn coordinating Cys residues of the Zn finger scaffold (i.e. the inter-cysteine loop). Zn coordinating Cys residues may be located in the Zn finger scaffold at positions

corresponding to residues 5 and 10 of SEQ ID NO: 423. A peptide ligand may be located between residues corresponding to 5 and 6, 6 and 7, 7 and 8, 8 and 9 or 9 and 10 of SEQ ID NO: 423. For example, a peptide ligand may be located in the Zn finger scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 5, 6, 7 or 8 of SEQ ID NO: 423. The peptide ligand may be inserted into the Zn finger scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 7, 8, 9, or 10 of SEQ ID NO: 423. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 6 to 9 of SEQ ID NO: 423.

A grafted Zn finger scaffold may contain a peptide ligand within the second loop, between Zn coordinating Cys and His residues of the Zn finger scaffold. A peptide ligand may be located between residues corresponding to 10 and 11 of SEQ ID NO: 423. For example, a peptide ligand may be located in the Zn finger scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 9 or 10 of SEQ ID NO: 423. The peptide ligand may be inserted into the Zn finger scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 11 or 12 of SEQ ID NO: 423. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the second loop. For example, a peptide ligand may replace the loop residues corresponding to residues 10 and 11 of SEQ ID NO: 423.

In some embodiments, a grafted Zn finger scaffold may comprise a first peptide ligand within the inter-cysteine loop. For example, a grafted Zn finger scaffold may comprise the amino acid sequence of SEQ ID NO: 425 or a variant thereof.

RPYACP [XiX ₂ ...x _n ] vESCDRRFSRSDELTRHiRiHTGQ (SEQ ID NO: 425 where [Xi X2... X _n ] is a peptide ligand of n amino acids, n is 3-30, typically 7-8; and Xi, X2...X _n are independently any amino acid are each independently any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.). In some embodiments, a grafted Zinc finger scaffold may contain a helical peptide ligand within the helical region located between residues corresponding to positions 17 to 28 of SEQ ID NO: 423.

A helical peptide ligand may be inserted in a helical portion of the Zn finger scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure). A“helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold. In some embodiments, a helical peptide ligand replaces the helix of the Zinc finger scaffold. For example, a peptide ligand may replace one or more of residues 17 to 28 of SEQ ID NO: 423. In some embodiments, a grafted Zinc finger scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27 of SEQ ID NO: 423. In some embodiments, a grafted Zinc finger scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 18, 19, 20, 21 , 22, 23, 24, 24, 25, 27, 28 or 29 of SEQ ID NO: 423). In some embodiments, a helical peptide ligand replaces the helix of the Zn finger scaffold. For example, a peptide ligand may replace residues corresponding to one or more of residues S17, D18, E19, T21 , R22, R25, I26 and T28 of SEQ ID NO: 423. Scaffold residues corresponding to L20 and I24, as well as residues corresponding to Zn coordinating residues H23 and H24 may not be replaced.

Suitable peptide ligands include helical peptide ligands as described herein. Suitable helical peptide ligands may be 12 to 30 amino acids in length.

In some embodiments, a grafted Zn scaffold is created by isomorphic replacement of those residues (for example in SEQ ID NO: 423 one or more of S17, D18, E19, T21 , R22, R25, 126 and T28) of the helix portion of the Zn scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.

For example, a helix peptide ligand may comprise the amino acid sequence of SEQ ID NO: 426, a fragment of SEQ ID NO: 426 or a variant of either of these. (SEQ ID NO: 426,_where Zi to Zs are independently any

amino acid any amino acid, for example an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine.)

In some preferred embodiments, a grafted Zinc finger scaffold may contain a helical peptide ligand in the helical region of the Zinc finger scaffold between positions corresponding to 17 and 28 of SEQ ID NO: 423 and a loop peptide ligand in one of (i) first loop of the Zinc finger scaffold between the positions corresponding to residues 6 and 9 of SEQ ID NO: 423 and (ii) the second loop between the positions corresponding to residues 11 and 12 of SEQ ID NO: 423.

The Zn finger scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Zn finger scaffolds.

Examples of grafted Zn finger scaffolds according to the twelfth aspect of the invention are shown in Figure 57B and Table 51 (SEQ ID NOs 802-810).

For example, a grafted Zn finger scaffold may comprise a Zn finger scaffold with the amino acid sequence of residues 1 to 42 of SEQ ID NO: 806 (PPX149 of Table 51 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 17-18 of SEQ ID NO: 806), second loop (loop 2, residues 20 to 21 of SEQ ID NO: 806) or the helix (helix 1 ; residues 22 to 30 of SEQ ID NO 806) of the Zn finger scaffold. Preferably, the peptide ligand is located in one of the first loop, second loop or helix. Preferably, the E3 ligase-binding peptide ligand is located in another of the first loop, second loop or helix. For example, the target-binding peptide ligand may be in the helix and the E3 ligase-binding peptide ligand may be in the first loop, the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop, or the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the helix.

Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE,

SEELEALEALELDEAS and GSEELEALEALELDEASGS and variants thereof, helical sequences from the protein AXIN, such as ILxxHV, AxxILDxHV, ILxxHVxxV, or ILDxHV and variants thereof, peptides from BCL9, such as TLxxlQxxL, LxTLxxlQ, and SLxxlxxML and variants thereof, helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE, TNxxLKxxE or IxxTNxxKTxE and variants thereof and peptides from KBL, such as PLYISY, PLYISYDPV and PLYISYPV and variants thereof. Suitable E3 ligase- binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLG, GLDPETGELLS and GLDPETGELL and variants thereof.

In some embodiments, a grafted Zn finger scaffold of the twelfth aspect may comprise an amino acid sequence shown in Table 51 or a variant of an amino acid sequence shown in Table 51. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 51 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the Zn finger scaffold sequence in a reference amino acid sequence of Table 51(SEQ ID NOs 801-809) is replaced by a different Zn finger scaffold sequence. Suitable Zn finger scaffold sequences are described above.

(xiii) SH3 Scaffolds

A thirteenth aspect of the invention relates to chimeric proteins that comprise SH3 scaffolds (i.e. grafted SH3 scaffolds). One or more peptide ligands are located in the SH3 scaffold of the chimeric protein, for example in the first, second, third and/or fourth loops of the SH3 scaffold.

The SH3 domain (SRC Homology 3 Domain) is a common binding domain found in multi- domain signalling proteins and other proteins.

The structure of the SH3 domain is well known in the art (see for example, Pfam PF00018, SMART 00326 (Meyer et al Journal of Cell Science. 114 (Pt 7): 1253-63; Schlatter D et al (2012), MAbs, 4:497; Filimonov F et al (1999), Biophys Chem, 77:195; Scwaher KL et al, (2007) Prot Sci, 16: 2694).

The invention adopts the well understood sequence-structure relationships of SH3 domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, an SH3 scaffold is a protein comprising a beta-barrel fold that consists of five to eight b-strands. The domains belong to the alpha+beta structural class, the beta- strands forming two tightly-packed, anti-parallel beta-sheets arranged in a barrel-like structure, and intervening loops sometimes forming helices. An SH3 scaffold as used herein may have a length in the amino acid range of 50 to 80 amino acids, for example, 56 and 76 amino acids, and any amino acid length there between, for example about 60 amino acids.

An SH3 scaffold may be in the MW range of 5500-9000 kDa, typically about 6980 kDa.

Suitable SH3 scaffolds useful according to the invention include SH3 domains selected from the following human proteins, without limitation: Rho GTPase-activating protein 10; Rho guanine nucleotide exchange factor 37; SH3 and PX domain-containing protein 2B; Rho GTPase-activating protein 42; RIMS-binding protein 3C; Rho GTPase-activating protein 32; Voltage-dependent L-type calcium channel subunit beta-4; Myc box-dependent-interacting protein 1 ; peripheral plasma membrane protein CASK; RIMS-binding protein 2;

nephrocystin-1 ; Kalirin; Periplakin; Vinexin; Growth arrest-specific protein 7; Src kinase- associated phosphoprotein 2; Tyrosine-protein kinase Lck; Tyrosine-protein kinase Fyn; Phosphatidylinositol 3-kinase regulatory subunit beta; Dynamin-binding protein and Proto oncogene tyrosine-protein kinase Src or variants of any one of these.

Suitable SH3 scaffolds include residues 85 to 140 of human Fyn (NP_694592.1) or variants thereof. Other suitable SH3 scaffold include the SH3 domains highlighted in Table 52 (SEQ ID NOs: 427 to 652) or variants thereof.

The following PDB codes from the Research Col laboratory for Structural Bioinformatics (RCSB) protein data bank (https://www.rcsb.org/; Berman et al P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242) identify representative structures of suitable SH3 scaffolds useful according to the invention without any limitation: 1 PKS, 2LMJ, 5NP3, 1WI7, 1 S1 N, 1YN2, 2HDA, p13K, 2KT1 , 1AON, 2JTG, 2KXD, 2XMF, ITG0, 1 UG1. (1ABO, 1ABQ, 1AD5, 1AEY, 1AOJ, 1ARK, 1AVZ, 1AWJ, 1AWO, 1AWW, 2A08, 2A28, 2A36, 2A37, 2ABL, 2AK5 and 1A0N). Other suitable Fyn3 SH3 structures in the PDB database are: 1A0N, 1A2G, 1 EFN, 1 G83, 1 M27, 1 NYF, 1 NYG, 1SHF, 12BJ, 6IPY, 3H0F, 3UA6, 4D8D, 4E1 K, 4ZNX, 6EDF, 6IP2.

Suitable SH3 scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44:D279-D285).

Other suitable scaffolds include In some embodiments, an SH3 scaffold may comprise the amino acid sequence of SEQ ID NO: 653 or a variant thereof.

The SH3 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 654 or a variant thereof.

(SEQ ID NO: 654; codons encoding loop residues underlined)

Preferred SH3 scaffolds lack lysine residues, for example, to avoid ubiquitination of the scaffold. There are no conserved lysine residues in the consensus SH3 sequence, and therefore any lysine residues in an SH3 domain may be replaced by either a polar or charged amino acid (D,E,H,K,N,Q,R,S,T) to generate a lysine- free SH3 scaffold. The SH3 domain of SEQ ID NO: 653 has two lysine residues (Lys23, Lys26), and these are both at solvent-exposed positions and could be changed for any polar or charged amino acids.

The first loop (R-loop) of an SH3 scaffold is located within the positions corresponding to residues 9 to 24 of SEQ ID NO: 653.

The second loop (Src loop) of an SH3 scaffold is located within the positions corresponding to residues 31 to 35 of SEQ ID NO: 653.

The third loop of an SH3 scaffold is located within the positions corresponding to residues 44 to 46 of SEQ ID NO: 653.

The fourth loop of an SH3 scaffold is located within the positions corresponding to residues 55 to 56 of SEQ ID NO: 653.

In some embodiments, an SH3 scaffold may comprise the amino acid sequence of SEQ ID NO: 655 or a variant thereof.

The SH3 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 656 or a variant thereof.

Preferred SH3 scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination of the scaffold. There are no conserved lysine residues in the consensus SH3 sequence, and therefore any lysine residues in an SH3 domain may be replaced by either a polar or charged amino acid (D,E,H,K,N,Q,R,S,T) to generate a lysine- free SH3 scaffold. The SH3 domain of SEQ ID NO: 655 has six lysine residues (Lys18, Lys46, Lys 47, Lys 48, Lys 65, Lys74) and these are all at solvent-exposed positions and could be changed for any polar or charged amino acids.

The first loop (R-loop) of an SH3 scaffold is located within the positions corresponding to residues 24 to 39 of SEQ ID NO: 655.

The second loop (Src loop) of an SH3 scaffold is located within the positions corresponding to residues 45 to 56 of SEQ ID NO: 655.

The third loop of an SH3 scaffold is located within the positions corresponding to residues 62 to 63 of SEQ ID NO: 655.

The fourth loop of an SH3 scaffold is located within the positions corresponding to residues 69 to 71 of SEQ ID NO: 655.

In some embodiments, an SH3 scaffold may contain an a helix. The a-helix is located within the positions corresponding to residues 4 to 13 of SEQ ID NO: 655.

In some embodiments, an SH3 scaffold may comprise a natural ligand interface for a target molecule, for example c-myc. A suitable SH3 scaffold may comprise the amino acid sequence of SEQ ID NO: 657 or a variant thereof.

GFMFKVQAQHDYTATDTDELQLKAGDWLVIPFQNPEEQDEGWLMGVKESDWNQHKKLEKC RGVFPENF

TERVP (SEQ ID NO: 657; loops underlined)

The SH3 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 658 or a variant thereof.

(SEQ ID NO: 658; loop codons underlined)

In some embodiments, an SH3 domain from a BAR protein, such as Endophilin A1 , may be employed. BAR protein SH3 domains bind to the E3 ubiquitin ligase Parkin. A suitable SH3 scaffold may comprise the amino acid sequence of SEQ ID NO: 659 or a variant thereof.

DQPCCRALYDFEPENEGELGFKEGDI ITLTNQIDENWYEGMLHGQSGFFPINYVEILVALPH

(SEQ ID NO: 659)

The SH3 scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 234 or a variant thereof.

Preferred SH3 scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, lysine residues that are present in an SH3 domain may be replaced by a polar or charged amino acid, such as D,E,H,K,N,Q,R,S,T, to generate a lysine-free SH3 scaffold. For example, the SH3 domain of SEQ ID NO: 657 has five lysine residues (Lys5, Lys47, Lys55, Lys56, Lys59) and these lysine residues are all at exposed positions and may be replaced by any polar or charged amino acid.

The first loop (R-loop) of an SH3 scaffold is located within the positions corresponding to residues 13 to 21 of SEQ ID NO: 657.

The second loop (Src loop) of an SH3 scaffold is located within the positions corresponding to residues 32 to 41 of SEQ ID NO: 657.

The third loop of an SH3 scaffold is located within the positions corresponding to residues 53 to 62 of SEQ ID NO: 657.

The fourth loop of an SH3 scaffold is located within the positions corresponding to residues 68 to 70 of SEQ ID NO: 657.

The positions of the loops are shown in the SH3 domain consensus sequences of Tables 53 and 54 together with the maximum number of amino acids in these loops for different members of the SH3 family (parenthesis). Peptide ligands may be located in one or more of the first, second, third and fourth loops in the SH3 scaffold, preferably one or more of the first, second and third loops in the SH3 scaffold. For example, peptide ligands may be located in the first and second loops in the SH3 scaffold.

A grafted SH3 scaffold contains peptide ligands in one, two or more loop portions of the SH3 scaffold, for example, inserted into a specific location of the loops (thus providing extended loops). The peptide ligand may be heterologous.

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the first loop (a“loop peptide”) i.e. the loop corresponding to residues 9 to 24 of SEQ ID NO: 653. Preferably, a peptide ligand is located between amino acids 10-24 of the first loop (to avoid disruption of a hydrogen bond between residues 9 and 25). For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,18, 19, 20, 21 , or 22 of SEQ ID NO: 653. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue

corresponding to residue 8, 9, 10, 11 ,12, 13, 14, 15, 16, 17,18, 19, 20, 21 , 22 or 23 of SEQ ID NO: 653. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 9 to 24 of SEQ ID NO: 653.

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the second loop (a“loop peptide”) i.e. the loop corresponding to residues 31 to 35 of SEQ ID NO: 653. Preferably, a peptide ligand is located between amino acids 32-34 of the second loop (to avoid disruption of a contacts between residues 31 and 30). .For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 30, 31 , 32, 33 or 34 of SEQ ID NO: 653. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 32, 33, 34, 35 or 36 of SEQ ID NO: 653. In some embodiments, the peptide ligand may replace the second loop. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 31 to 35 of SEQ ID NO: 653.

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the third loop (a“loop peptide”) i.e. the loop corresponding to residues 44 to 46 of SEQ ID NO: 653. Preferably, a peptide ligand is located between amino acids corresponding to residues 44 to 45 of the third loop (to avoid disruption of a contacts between residues 46 and 47). For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 43, 44 or 45 of SEQ ID NO: 653. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 45, 46 or 47 of SEQ ID NO: 653.

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the fourth loop (a“loop peptide”) i.e. the loop corresponding to residues 55 to 56 of SEQ ID NO: 653. Preferably, a peptide ligand is located between amino acids corresponding to residues 54 to 55 or 55-56 of the fourth loop. For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 54 or 55 of SEQ ID NO: 653. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 56 or 57 of SEQ ID NO: 653.

In some preferred embodiments, a grafted SH3 scaffold may comprise a peptide ligand within the first and second loops (the“R loop” and the“Src loop”).

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the first loop (a“loop peptide”) i.e. the loop corresponding to residues 24 to 39 of SEQ ID NO: 655. Preferably, a peptide ligand is located between amino acids corresponding to residues 24 to 31 of the first loop, or amino acids corresponding to residues 27 to 32. .For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 or 39 of SEQ ID NO: 655. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 of SEQ ID NO: 655. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the second loop (a“loop peptide”) i.e. the loop corresponding to residues 45 to 56 of SEQ ID NO: 655. Preferably, the peptide ligand is located between amino acids corresponding to residues 46 to 55 of the second loop. For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54 or 55 of SEQ ID NO: 655. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56 or 57 of SEQ ID NO: 655. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the third loop (a“loop peptide”) i.e. the loop corresponding to residues 62 to 63 of SEQ ID NO: 655. For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 61 or 62 of SEQ ID NO: 655. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 63 or 64 of SEQ ID NO: 655.

In some embodiments, a grafted SH3 scaffold may contain a peptide ligand within the fourth loop (a“loop peptide”) i.e. the loop corresponding to residues 69 to 71 of SEQ ID NO: 655. For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 68, 69 or 70 of SEQ ID NO: 653. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 70, 71 or 72 of SEQ ID NO: 655. The peptide ligand may be added to the fourth loop or may replace one or more residues of the fourth loop.

In some embodiments, a grafted SH3 scaffold may comprise a peptide ligand within a helical region, for example helical region at positions corresponding to residues 4 to 13 of SEQ ID NO: 655. A grafted SH3 scaffold may be created by isomorphic replacement of solvent exposed residues (for example 4, 5, 8, 9, 12 and 13 of SEQ ID NO: 655) of the helix (which are not buried in the hydrophobic core of the scaffold) with solvent facing residues of a donor ligand sequence. For example, a grafted SH3 scaffold may comprise the peptide ligand;

X _I ¾SGX ¾LLAX X PP (SEQ ID NO: 232 where X1 to X6 are independently any amino acid)

In some embodiments, a peptide ligand may be longer than the helical region of the SH3 scaffold, such that the N terminus of the grafted SH3 scaffold is extended.

In some preferred embodiments, a grafted SH3 scaffold may comprise a peptide ligand within the first or second loops (the“R loop” or the“Src loop”) and a peptide ligand grafted on to the outer face of the helix.

In some embodiments, an SH3 scaffold may comprise a natural ligand interface for a target molecule. An SH3 scaffold of SEQ ID NO: 657 may bind to c-myc. Interaction of the SH3 scaffold with the target molecule may hinder the interactions of peptide ligands located in loops 1 , 2 and 4 of the SH3 scaffold. A grafted SH3 scaffold comprising a natural ligand interface for a target molecule may comprise a peptide ligand within the third loop (a“loop peptide”) i.e. the loop corresponding to residues 51 to 61 of SEQ ID NO: 657. Preferably the peptide ligand is located between the residues corresponding to residues 52 to 56 of SEQ ID NO: 131. For example, a peptide ligand may be located in the SH3 scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 of SEQ ID NO: 657. The peptide ligand may be inserted into the SH3 scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 of SEQ ID NO: 657. The peptide ligand may be added to the third loop or may replace one or more residues of the third loop.

The SH3 scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the SH3 scaffolds.

In other embodiments, an SH3 scaffold of a chimeric protein may also display binding activity i.e. the SH3 scaffold may mediate binding of the chimeric protein to a first target molecule, such as c-myc or an E3 ubiquitin ligase. The peptide ligand may mediate binding of the chimeric protein to a second target molecule. For example, the SH3 scaffold of SEQ ID NO: 657 may bind to c-myc and the SH3 scaffold of SEQ ID NO: 659 may bind to the E3 ubiquitin ligase Parkin.

Examples of grafted SH3 scaffolds according to the thirteenth aspect of the invention are shown in Figure 62C and Table 55 (SEQ ID NOs 811-839).

For example, a grafted SH3 scaffold may comprise an SH3 scaffold with the amino acid sequence of residues 1 to 97 of SEQ ID NO: 811 (PPX35 of Table 55 without the HA Tag). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 34-35 of SEQ ID NO: 811), second loop (loop 2, residues 70 to 71 of SEQ ID NO: 811) or the helix (helix 1 ; residues 7 to 17 of SEQ ID NO 811) of the SH3 scaffold.

Preferably, the peptide ligand is located in one of the first loop and the helix of the SH3 scaffold. Preferably, the E3 ligase-binding peptide ligand is located in the other of the first loop and the helix. For example, the target-binding peptide ligand may be in the helix and the E3 ligase-binding peptide ligand may be in the first loop, and; the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the helix.

In other embodiments, a grafted SH3 scaffold may comprise an SH3 scaffold with the amino acid sequence of residues 84 to 141 of UniProt KB P06241 (FYN_HUMAN). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into the first loop (loop 1 ; residues 116-118 of UniProt KB P06241) or the second loop (loop 2, residues 126- 127 of UniProt KB P06241). Preferably, the peptide ligand is located in the first loop of the SH3 scaffold. Preferably, the E3 ligase-binding peptide ligand is located in the second loop of the SH3 scaffold.

In some embodiments, a grafted SH3 scaffold of the thirteenth aspect may comprise an amino acid sequence shown in Table 55 or a variant of an amino acid sequence shown in Table 55. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 55 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the SH3 scaffold sequence in a reference amino acid sequence of Table 55 (SEQ ID NOs 810-839) is replaced by a different SH3 scaffold sequence. Suitable SH3 scaffold sequences are described above.

(xiv) Cystine knot (CK) scaffolds A fourteenth aspect of the invention relates to chimeric proteins that comprise cystine knot scaffolds (i.e. grafted cystine knot scaffolds). One or more peptide ligands are located in the cystine knot scaffold of the chimeric protein, for example in the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold.

A cystine knot domain is a protein structural motif comprising a knotted arrangement of three interlinked intra-molecular disulphide linkages between cysteine residues I and IV, II and V and III and VI. A cysteine knot domain may be an Inhibitor Cystine Knot domain (ICK or knottin) comprising free N- and C-termini or a Cyclic Cystine Knot domain (CCK) having Island C-termini linked by a peptide bond (i.e. a closed intramolecular ring structure). The use of CK domains in protein engineering is established in the art (see for example Kintzing et al (2016) Curr Opin Chem Biol 34 143-150; Poth et al (2013) Peptide Science 100 5 480-491 ; de Veer et al (2017) Acc Chem Res 50 1557-1565).

The structures of CK domains, for example ICK domains (see for example PFAM 17486; Chen J, et al, Cell Mol Life Sci. 2008;65:2431-2444; Windley MJ, et al;, Toxins (Basel).

2012;4:191-227) and CCK domains (see for example PFAM03784; Daly NL et al, J Mol Biol 1999;285:333-345; Craik DJ, et al J Mol Biol 1999;294:1327-1336), are well known in the art. These domains sufficiently small to be synthetically tractable (hence allowing incorporation of unnatural amino acids) as well as genetically encodable.

The invention adopts the well understood sequence-structure relationships of CK domains, such as CCK and ICK domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.

As used herein, a CK scaffold is folded peptide comprising a loop formed by intra-molecular disulphide linkages between cysteine residues I and IV and II and V. A third intra-molecular disulphide linkage between cysteine residues III and VI passes through this loop to form a knot (i.e. a CK domain structure). A CK scaffold may have free N- and C-termini (ICK scaffold) or the N- and C-termini may be connected through a peptide bond to form a cyclic structure (CCK domain).

A CK scaffold, as used herein, has a length in the amino acid range of 20 to 40 amino acids, for example 28 to 37 amino acids, and any amino acid length there between, for example about 32 amino acids. A CK scaffold may be in MW range of 2 kDa to 4.5 kDa, for example 3 kDa to 4 kDa, typically about 3.5 kDa.

Suitable CK scaffolds useful according to the invention include the ICK domains selected from the following, without limitation: Ecballium elaterium trypsin inhibitor II (EETII), MCh-1 , vanillotoxin, tachystatin, strom atoxin, robustoxin, psalmotoxin, phrixotoxin, d-palutoxin, theraphosa leblondi toxin, maurocalcine, and huwentoxin; and the CCK domains selected from the following, without limitation: MCoTI-l, MCoTI-ll, cycloviolacin 012, kalata B1 , varv peptide A, tricyclon A, palicourein, kalata B8, cycloviolacin 01 , circulin A, cyclopsychotride A or variants of any one of these.

The following PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable CCK scaffolds useful according to the invention without any limitation: 1 NB1 , 1 IB9, 2KNM, 2ERI, 1 KAL, 1 BH4, 1 DF4, 1 HA9, 2F2I and 1J7.

The following PDB codes from the protein data bank identify representative structures of suitable ICK scaffolds useful according to the invention without any limitation: 2IT7, 2M2Q, 2C4B, 2M2Q, 2M2R, 6ATU, 2N8E, 1 MR0, 1 KOZ, and 1 IB9.

Suitable CK scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44:D279-D285).

In some embodiments, a CK scaffold may be a CCK scaffold comprising the amino acid sequence of SEQ ID NO: 840 or a variant thereof.

(SEQ ID NO: 840; disulfide forming cysteines (I

to VI; numbered sequentially) underlined)

The CCK scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 841 or a variant thereof.

In some embodiments, a CK scaffold may be a CCK scaffold comprising the amino acid sequence of SEQ ID NO: 842 or a variant thereof. (SEQ ID NO: 842; disulfide forming cysteines (I to VI) underlined)

The CK scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 843 or a variant thereof.

In some embodiments, a CK scaffold may be a CCK scaffold comprising the amino acid sequence of SEQ ID NO: 844 or a variant thereof. cpKILKKCRRDsD cpGACiCRGNGYCGsGsDGGv (SEQ ID NO: 844; disulfide forming cysteines (I to VI) underlined)

The CK scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 845 or a variant thereof.

A CCK scaffold may be cyclic i.e. the N terminus and C terminus residues may be linked by a peptide bond.

In some embodiments, a CK scaffold may be an ICK scaffold comprising the amino acid sequence of SEQ ID NO: 846 or a variant thereof.

GCPRILMRCKQD s DCLAGCvcGPNGFCG sp (SEQ ID NO: 846; disulfide forming cysteines (I to VI) underlined)

The ICK scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 847 or a variant thereof.

In some embodiments, a CK scaffold may be an ICK scaffold comprising the amino acid sequence of SEQ ID NO: 848 or a variant thereof. (SEQ ID NO: 848; disulfide forming cysteines (I to VI sequentially are underlined)

The ICK scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 849 or a variant thereof. Preferred CK scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination. For example, the lysine residues in a CK domain may be replaced by either a polar or charged amino acid (D,E,H,K,N,Q,R,S,T) to generate a lysine- free CK scaffold. A suitable lysine-free CK scaffold may comprise the amino acid sequence of SEQ ID NO: 840 or a variant thereof.

The first loop of a CK scaffold is located between cysteines I and II, for example at positions corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842, residues 2 to 7 of SEQ ID NO: 844; residues 3 to 8 of SEQ ID NO: 846 and residues 3 to 4 of SEQ ID NO:

848.

The second loop of a CK scaffold is located between cysteines II and III, for example at positions corresponding to residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 3, residues 9 to 13 of SEQ ID NO: 844; residues 10 to 14 of SEQ ID NO: 846 and residues 6 to 15 of SEQ ID NO: 848.

The third loop of a CK scaffold is located between cysteines III and IV, for example at a position corresponding to residues 11 to 14 of SEQ ID NO: 840; residues 11 to 16 of SEQ ID NO: 3, residues 15 to 17 of SEQ ID NO: 844; residues 16 to 18 of SEQ ID NO: 846 and residues 17 to 19 of SEQ ID NO: 848.

The fourth loop of a CK scaffold is located between cysteines IV and V, for example at a position corresponding to residue 16 of SEQ ID NO: 840; residue 18 of SEQ ID NO: 3, residue 19 of SEQ ID NO: 844; residue 20 of SEQ ID NO: 846 and residues 21 to 27 of SEQ ID NO: 848.

The fifth loop of a CK scaffold is located between cysteines IV and V, for example at positions corresponding to residues 18 to 21 of SEQ ID NO: 840; residues 20 to 23 of SEQ ID NO: 3, residues 21 to 25 of SEQ ID NO: 844; residues 22 to 26 of SEQ ID NO: 846 and residues 29 to 32 of SEQ ID NO: 848.

The sixth loop of a CK scaffold is located between cysteines V and V1 , for example at positions corresponding to residues 23 to 30 of SEQ ID NO: 840; residues 25 to 31 of SEQ ID NO: 3, residues 27 to 35 of SEQ ID NO: 844; residues 28 to 30 of SEQ ID NO: 846 and residue 34 of SEQ ID NO: 848.

A grafted CK scaffold may comprise peptide ligands in two or more of the first, second, third, fourth, fifth and sixth loops of the CK scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). The peptide ligand may be heterologous.

A grafted CK scaffold may comprise a peptide ligand in the first loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 1 , 2 or 3 of SEQ ID NOs: 1 or 3, residues 1 , 2, 3, 4, 5 or 6 of SEQ ID NO: 844, residues 2, 3, 4, 5, 6 or 7 of SEQ ID NO: 846 or residues 2 or 3 of SEQ ID No 9. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue corresponding to residue 3, 4 or 5 of SEQ ID NOs: 1 or 3, residues 3, 4, 5, 6, 7 or 8 of SEQ ID NO: 844, residues 4, 5, 6, 7, 8 or 9 of SEQ ID NO: 846 or residues 3 or 4 of SEQ ID No 9. The peptide ligand may be added to the first loop or may replace one or more residues of the first loop. For example, the peptide ligand may replace the first loop. For example, a peptide ligand may replace the loop residues corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 3, residues 2 to 7 of SEQ ID NO: 844; residues 3 to 8 of SEQ ID NO: 846 and residues 3 to 4 of SEQ ID NO: 848.

A grafted CK scaffold may comprise a peptide ligand in the second loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 5, 6, 7 or 8 of SEQ ID NOs: 1 or 3, residues 8, 9, 10, 11 or 12 of SEQ ID NO: 844, residues 9, 10, 11 , 12 or 13 of SEQ ID NO: 846 or residues 5, 6, 7, 8, 9,

10, 11 , 12, 13 or 14 of SEQ ID NO: 848. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue 7, 8, 9 or 10 of SEQ ID NOs: 1 or 3, residues 10, 11 , 12, 13 or 14 of SEQ ID NO: 844, residues 11 , 12, 13, 14 or 15 of SEQ ID NO: 846 or residues 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 of SEQ ID NO: 848. The peptide ligand may be added to the second loop or may replace one or more residues of the second loop. For example, the peptide ligand may replace the second loop. For example, a peptide ligand may replace the loop residues corresponding to residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 3, residues 9 to 13 of SEQ ID NO: 844; residues 10 to 14 of SEQ ID NO: 846 and residues 6 to 15 of SEQ ID NO: 848.

A grafted CK scaffold may comprise a peptide ligand in the third loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residues 10, 11 , 12 or 13 of SEQ ID NO: 840, residues 10, 11 , 12, 13, 14 or 15 of SEQ ID NO: 3, residues 14, 15 or 16 of SEQ ID NO: 844, residues 15, 16 or 17 of SEQ ID NO: 846 or residues 16, 17 or 18 of SEQ ID No 9. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue corresponding to residues 12, 13, 14 or 15 of SEQ ID NO: 840, residues 12, 13, 14, 15, 16 or 17 of SEQ ID NO: 3, residues 16, 17 or 18 of SEQ ID NO: 844, residues 17, 18 or 19 of SEQ ID NO: 846 or residues 17, 18 or 20 of SEQ ID NO: 848. The peptide ligand may be added to the third loop or may replace one or more residues of the third loop. For example, the peptide ligand may replace the third loop. For example, a peptide ligand may replace the loop residues corresponding to residues 11 to 14 of SEQ ID NO: 840; residues 11 to 16 of SEQ ID NO: 3, residues 15 to 17 of SEQ ID NO: 844; residues 16 to 18 of SEQ ID NO: 846 and residues 17 to 19 of SEQ ID NO: 848.

A grafted CK scaffold may comprise a peptide ligand in the fourth loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residue 15 of SEQ ID NO: 840, residue 17 of SEQ ID NO: 3, residue 18 of SEQ ID NO: 844, residue 19 of SEQ ID NO: 846 or residues 20, 21 , 22, 23, 24, 25 or 26 of SEQ ID No 9. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue corresponding to residue 17 of SEQ ID NO: 840, residue 19 of SEQ ID NO: 3, residue 20 of SEQ ID NO: 844, residue 21 of SEQ ID NO: 846 or residues 22, 23, 24, 25, 26, 27 or 28 of SEQ ID NO: 848. The peptide ligand may be added to the fourth loop or may replace one or more residues of the fourth loop. For example, the peptide ligand may replace the fourth loop. For example, a peptide ligand may replace the loop residues corresponding to residue 16 of SEQ ID NO: 840; residue 18 of SEQ ID NO: 3, residue 19 of SEQ ID NO: 844; residue 20 of SEQ ID NO: 846 and residues 21 to 27 of SEQ ID NO: 848.

A grafted CK scaffold may comprise a peptide ligand in the fifth loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residues 17, 18, 19 or 20 of SEQ ID NO: 840, residues 19, 20, 21 or 22 of SEQ ID NO: 3, residues 20, 21 , 22, 23 or 24 of SEQ ID NO: 844, residues 21 , 22, 23, 24 or 25 of SEQ ID NO: 846 or residues 28, 29, 30 or 31 of SEQ ID NO: 848. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue corresponding to residues 19, 20, 21 or 22 of SEQ ID NO: 840, residues 21 , 22, 23 or 24 of SEQ ID NO: 3, residues 22, 23, 24, 25 or 26 of SEQ ID NO: 844, residues 23, 24, 25, 26 or 27 of SEQ ID NO: 846 or residues 20, 31 , 32 or 33 of SEQ ID NO: 848. The peptide ligand may be added to the fifth loop or may replace one or more residues of the fifth loop. For example, the peptide ligand may replace the fifth loop. For example, a peptide ligand may replace the loop residues corresponding to residues 23 to 30 of SEQ ID NO: 840; residues 25 to 31 of SEQ ID NO: 3, residues 27 to 35 of SEQ ID NO: 844; residues 28 to 30 of SEQ ID NO: 846 and residue 34 of SEQ ID NO: 848.

A grafted CK scaffold may comprise a peptide ligand in the sixth loop. For example, a peptide ligand may be located in the CK scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to residues 22, 23, 24, 25, 26, 27, 28 or 29 of SEQ ID NO: 840, residues 24, 25, 26, 27, 28, 29 or 30 of SEQ ID NO: 3, residues 26, 27, 28, 29, 30, 31 , 32, 33 or 34 of SEQ ID NO: 844, residues 27, 28 or 29 of SEQ ID NO: 846 or residue 33 of SEQ ID No 9. The peptide ligand may be inserted into the CK scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to the residue corresponding to residues 24, 25, 26, 27, 28, 29, 30 or 31 of SEQ ID NO: 840, residues 26, 27, 28, 29, 30, 31 or 32 of SEQ ID NO: 3, residues 28, 29, 30, 31 , 32, 33, 34, 35 or 36 of SEQ ID NO: 844, residues 29, 30 or 32 of SEQ ID NO: 846 or residue 35 of SEQ ID NO: 848. The peptide ligand may be added to the sixth loop or may replace one or more residues of the sixth loop. For example, the peptide ligand may replace the sixth loop. For example, a peptide ligand may replace the loop residues corresponding to residues 18 to 21 of SEQ ID NO: 840; residues 20 to 23 of SEQ ID NO: 3, residues 21 to 25 of SEQ ID NO: 844; residues 22 to 26 of SEQ ID NO: 846 and residues 29 to 32 of SEQ ID NO: 848.

Suitable peptide ligands may be 3 to 50 amino acids, for example 5 to 30 amino acids. In some embodiments, the peptide ligands may be non-helical in structure or unstructured.

A grafted CK scaffold may comprise;

a first peptide ligand in the first loop and a second peptide ligand in the second loop, a first peptide ligand in the first loop and a second peptide ligand in the third loop, a first peptide ligand in the first loop and a second peptide ligand in the fourth loop, a first peptide ligand in the first loop and a second peptide ligand in the fifth loop, a first peptide ligand in the first loop and a second peptide ligand in the sixth loop, a first peptide ligand in the second loop and a second peptide ligand in the third loop, a first peptide ligand in the second loop and a second peptide ligand in the fourth loop,

a first peptide ligand in the second loop and a second peptide ligand in the fifth loop, a first peptide ligand in the second loop and a second peptide ligand in the sixth loop, a first peptide ligand in the third loop and a second peptide ligand in the fourth loop, a first peptide ligand in the third loop and a second peptide ligand in the fifth loop, a first peptide ligand in the third loop and a second peptide ligand in the sixth loop, a first peptide ligand in the fourth loop and a second peptide ligand in the fifth loop, a first peptide ligand in the fourth loop and a second peptide ligand in the sixth loop; or a first peptide ligand in the fifth loop and a second peptide ligand in the sixth loop

Preferred grafted CK scaffolds may comprise;

a first peptide ligand in the second loop and a second peptide ligand in the sixth loop a first peptide ligand in the third loop and a second peptide ligand in the sixth loop, a first peptide ligand in the fifth loop and a second peptide ligand in the sixth loop; or a first peptide ligand in the third loop and a second peptide ligand in the fifth loop.

For example, a CK scaffold may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands. For example, aromatic residues in the CK scaffold may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of CK scaffold sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.

In some embodiments, lysine residues in the CK scaffold may be replaced by a different residue, such as arginine to prevent unwanted ubiquitination and subsequent degradation. This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase- peptide ligand, for example in a proteolysis targeting chimera (PROTAC).

Examples of grafted CK scaffolds according to the fourteenth aspect of the invention are shown in Figure 68 and Table 56 (SEQ ID NOs: 852-855).

For example, a grafted CK scaffold may comprise a CK scaffold with the amino acid sequence of of SEQ ID NO: 855 (see Table 56). A target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of first loop (loop 1 ; residues 5 to 10 of SEQ ID NO: 855), second loop (loop 2; residues 12 to 16 of SEQ ID NO: 855), third loop (loop 3; residues 18 to 20 of SEQ ID NO: 702), fourth loop (loop 4; residues 22 of SEQ ID NO: 855), fifth loop (loop 5; residued 24 to 28 of SEQ ID NO: 855) and sixth loop (loop 6; residues 30 to 34 of SEQ ID NO: 855) of the CK scaffold. Preferably, the target-binding peptide ligand is located in the first, five or sixth loop. Preferably, the E3 ligase-binding peptide ligand is located in the sixth, or fifth loop. For example, the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the sixth loop; the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the fifth loop or the target-binding peptide ligand may be in the sixth loop and the E3 ligase-binding peptide ligand may be in the first loop. Suitable target binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE or GSEELEALEALELDEA and variants thereof, and KRAS binding ligands, for example peptides from KBL, such as PLYISY, PLYISYDPV and PLYISYPV and variants thereof. Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGELLG variants thereof and MDM2 binding sequences from p53, such as FSxxWxxL, FxxxWxxL and variants thereof.

In some embodiments, a grafted CK scaffold of the fourteenth aspect may comprise an amino acid sequence shown in Table 56 (SEQ ID NOs: 852-855) or a variant of an amino acid sequence shown in Table 56. Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 56 are replaced by a different peptide ligand. Suitable peptide ligands are described below. Variants may also include variants in which the CK scaffold sequence in a reference amino acid sequence of Table 56 is replaced by a different CK scaffold sequence. Suitable CK scaffold sequences are described above.

The CK scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the CK scaffolds.

Peptide Ligands

A peptide ligand is a contiguous amino acid sequence that specifically binds to a target molecule. A peptide ligand may be grafted onto a helical region or loop region of the scaffold. Suitable peptide ligands are well-known in the art and include peptide sequences selected from a library, antigen epitopes, natural protein-protein interactions (helical, extended or turn-like) and short linear motifs (SLiMs). Viral SLiMs (that hijack the host machinery) may be particularly useful because they may display high binding affinities (Davey et al (2011) Trends Biochem. Sci. 36,159-169). Suitable peptide ligands that bind to different targets are described herein.

Peptide ligands may be 3 to 30 or 3 to 25 amino acids in length, preferably 8 to 15 amino acids, although in some embodiments, longer peptide ligands up to 50 amino acids may be employed. Peptide ligands may comprise 3-10 or 3-12 or 3-15 or 8-10 or 8-12 or 8-13 or 8- 14 or 8-15 or 3-18 or 3-20 or 3-21 or 3-22 or 3-24 or 3-25 amino acids.

In some embodiments, the peptide ligands and the scaffold of the chimeric protein of any one of the first to the fourteenth aspects are heterologous i.e. the peptide ligand is not associated with the scaffold in naturally occurring proteins. The peptide ligand and scaffold are artificially associated in the chimeric protein by recombinant means. In other

embodiments, the scaffold of the chimeric protein may have a natural binding affinity, for example to a target molecule an E3 ligase (e.g. CKS1 binds to the E3 ligase SCF ^Skp2 ) or the proteasome (e.g. ubiquitin-like domain, such as PLIC2 may bind to the Rpn13 subunit of the proteasome). A first peptide ligand may be heterologous to the scaffold of the chimeric protein and a second peptide ligand may be endogenous or naturally occurring in the scaffold.

A suitable peptide ligand for a target molecule may be selected from a library, for example using phage or ribosome display, or identified or designed using rational approaches or computational design, for example using the crystal structure of a complex or an interaction. In some embodiments, peptide ligands may be identified in an amino acid sequence using standard sequence analysis tools (e.g. Davey et al Nucleic Acids Res. 2011 Jul 1 ; 39 (Web Server issue): W56-W60).

A chimeric protein described herein may comprise 1 , 2 or more than 3 peptide ligands. Preferably, the chimeric protein comprises 2 or more peptide ligands. The number of peptide ligands is determined by the required functionality and valency of the chimeric protein and the available loops and helices of the scaffold. For example, one peptide ligand may be suitable for a mono-functional chimeric protein and two or more peptide ligands may be suitable for a bi-functional or multi-functional chimeric protein.

Chimeric proteins may be monovalent. A target molecule may be bound by a single peptide ligand in a monovalent chimeric protein. Chimeric proteins may be multivalent. A target molecule may be bound by two or more of the same or different peptide ligands in a multivalent chimeric protein. Chimeric proteins may be monospecific. The peptide ligands in a monospecific chimeric protein may all bind to the same target molecule, more preferably the same site or epitope of the target molecule.

Preferably, chimeric proteins are multi-specific. The peptide ligands in a multi-specific chimeric protein may bind to different target molecules. For example, a bispecific chimeric protein may comprise a peptide ligand that binds to a first target molecule and a peptide ligand that binds to a second target molecule and a trispecific chimeric protein may comprise a peptide ligand that binds to a first target molecule, a peptide ligand that binds to a second target molecule and a peptide ligand that binds to a third target molecule.

A bispecific or hetero-bifunctional protein may bind to the two different target molecules concurrently. This may be useful in bringing the first and second target molecules into close proximity. When the target molecules are located on different cells, concurrent binding of the target molecules to the chimeric protein may bring the cells into close proximity, for example to promote or enhance the interaction of the cells. For example, a chimeric protein which binds to a tumour specific antigen and a T cell antigen, such as CD3, may be useful in bringing T cells into proximity to tumour cells. When the target molecules are from different biological pathways, this may be useful in achieving synergistic effects and also for minimising resistance. In some preferred embodiments, the one of the first and second target molecules may be a member of a component degradation pathway, such as an E3 ubiquitin ligase. The chimeric protein may bring the other of the one of the first and second target molecules into proximity with the member of a cellular degradation pathway, thereby promoting degradation of the target molecule.

A trispecific chimeric protein may bind to three different target molecules concurrently. In some embodiments, one of the target molecules may be a component of a cellular degradation pathway, such as an E3 ubiquitin ligase. For example, tri-specific chimeric protein may bind to a first target molecule from a first biological pathway and a second target molecule from a second biological pathway as well as an E3 ubiquitin ligase. This may be useful in achieving synergistic effects and also for minimising resistance.

A peptide ligand may be located in a loop region of the scaffold in the chimeric protein of the first to the fourteenth aspects. A loop region or“loop” is a portion of a scaffold which is not structured in that it does not assume an a-helix, a coil, a b-sheet, or any other ordered component of tertiary structure. Typically, a loop region may be about 3 to 15 residues in length. The loop region may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids long.

A loop peptide ligand may be shorter than the loop region or the same length as the loop region of a scaffold of the first to the fourteenth aspects. However, since there is no intrinsic restriction on the size of the loop peptide ligand, longer sequences of more than 25 amino acid residues may be used in some embodiments.

In some embodiments, an unstructured peptide ligand may be inserted into a loop.

A peptide ligand may be connected to a loop region of a scaffold of the first to the fourteenth aspects directly or via one or more additional residues or linkers. Additional residues or linkers may be useful for example when a peptide ligand requires conformational flexibility in order to bind to a target molecule, or when the amino acid residues that are adjacent to the minimal peptide ligand favourably influence the micro-environment of the binding interface.

Additional residues or linkers may be positioned at the N terminus of the peptide ligand, the C terminus of the peptide ligand, or both. For example, the sequence of a loop containing a peptide ligand may be [Xi- _/ ]-[Xi- _n ]-[Xi- _z ], where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue that is also denoted by X, [Xi. _n ] is the peptide ligand, n is 1 to 100, [Xi_,] is a linker and / is independently any number between 1 to 10.

The precise sequence of the residues or linkers used to connect a peptide ligand to a loop depends on the peptide ligand and may be readily determined for any peptide ligand of interest using standard protein engineering concepts. For example, small, non-hydrophobic amino acids, such as glycine, may be used to provide flexibility and increased spatial sampling, for example when a peptide ligand needs to adopt a specific conformation, or proline residues may be used to increase rigidity, for example, when the peptide ligands are short.

In some preferred embodiments, a loop peptide ligand may be non-hydrophobic. For example, at least 40% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W). Alternatively, the scaffolds of the first to the fourteenth aspects may be modified to accommodate a hydrophobic peptide ligand, for example by replacing aromatic residues with charged or polar residues.

A peptide ligand may be located in a helical region of the scaffold in a chimeric protein of the first to the fourteenth aspects. A helical region or“helix” is a portion of a scaffold which assumes an a-helical structure.

In some embodiments the length of the helical peptide is determined by the length of the helix of the scaffold to which the helical peptide ligand is grafted. In some embodiments, the length of the helical peptide is equal to or less than the length of the helix of the scaffold in which grafting occurs

The precise length of a helical peptide ligand is dependent on the length of the helical region of the scaffold. In general, the helical peptide ligand is no longer than the length of the helical region of the scaffold. However, if the helical region of the scaffold is located at one or other termini or is flanked by unstructured or loosely structured residues, then it may be possible to extend it to accommodate a longer helical peptide ligand.

A helical peptide ligand may comprise 3 to 30, preferably 3 to 25 amino acid residues, more preferably 8 to 15 amino acids in length. In some embodiments, a helical peptide ligand may comprise 3-10 or 3-12 or 3-15 or 8-10 or 8-12 or 8-13 or 8-14 or 8-15 or 3-18 or 3-20 or 3-21 or 3-22 or 3-24 or 3-25 amino acids. In some embodiments, a helical peptide ligand may comprise 3 or 4 or 5 or 6 or 7 or8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 amino acid residues.

A helical peptide ligand may comprise an a-helical structure and may comprise all or part of a half-repeat (i.e. all or part of a single a-helix) of the scaffold. The a-helix of the helical peptide ligand makes stabilising interactions with the scaffold and is stable and folded. Only a few of the positions that structurally define an a-helix are required for the correct interfacial interaction with the scaffold. The residues in some of these positions are defined and unmodified but the remaining positions of the a-helix may be modified to form a helical peptide ligand.

For example, in a coiled-coil scaffold of the second aspect with the amino acid sequence of SEQ ID NO: 1 the following amino acids form the hydrophobic would not be changed in helix 1 V19, N20, L23, E28, R29, L31 , E32, 134, E37, L38, K41 , A44, E45, R48, T51 and in helix 2 V61 , L65, S68, S69, L72, L74, L75, Q77, L79, L82. In a Ubiquitin scaffold of the sixth aspect, the residues I23, V26, and 130 are defined but the remaining positions of the a-helix may be modified to form a helical peptide ligand. In a GB1 scaffold of the seventh aspect, the residues A22, A25, F30 and A33 are defined but the remaining positions of the a-helix may be modified to form a helical peptide ligand. It is within the skill in the art to graft selected residues of a peptide ligand into a helix portion of a scaffold containing a helix, and the invention contemplates this variation of grafting as an equivalent to grafting a peptide ligand itself. The residues of the peptide ligand that are in contact with the peptide ligand binding partner (the target protein) are those whose side chains are outward facing and are exposed to solvent. These residues are suitable for grafting to a helical portion of a scaffold. The residues of the scaffold helix whose side chains face inwards and pack against the rest of the scaffold should not be substituted, and this way their interactions with the rest of the scaffold are maintained. It is within the skill in the art to visualize the scaffold structure to identify which of the residues of the helix selected for grafting are facing outwards. PDB codes from any protein databank provide three

dimensional co-ordinates that allow one of skill in the art to visualize the structure of the domain using programs such as PYMOL®, CHIMERA® and RASMOL®. At the same time, it is well within the skill in the art to identify residues of the helix peptide ligand that face outwards form noncovalent interactions (hydrogen bonds and/or Van de Waals and/or hydrophobic interactions) with its binding partner, using a program such as PYMOL®, CHIMERA® and RASMOL® to visualize a peptide ligand complexed with its binding partner. Helix grafting is performed by selectively replacing the outward-facing residues of the helix with corresponding outward-facing residues of the peptide ligand. The inward-facing residues of the helix are undisturbed, and hence the resultant grafted scaffold will have a grafted helix that comprises a mixture of outward facing residues derived from the helix peptide and the native inward facing residues of the helix that were undisturbed.

For instance, the following example shows a nine-residue helix peptide ligand (X1-X2-X3- X4-X5-X6-X7-X8-X9) . A 3-dimensional view of the peptide ligand in complex with the target protein (using one of the above-noted programs) shows that residues X1 , X2, X5, X8 and X9 (for example) of the peptide ligand interact with the target protein and thus are outward facing. Similarly, a helical portion of a given scaffold may be thirty amino acids in length (Y1-

Y2-Y3- . -Y28-Y29-Y30). A 3-dimensional view of the scaffold shows the helical region and that residues Y3, Y4, Y6, Y7, and Y10 (for example) are inward facing and thus interact with the rest of the scaffold. One of skill in the art would recognize Y1 , Y2, Y5, Y8 and Y9 as outward facing, thus identifying these residues as scaffold helical residues that may be replaced with peptide ligand outward facing residues. Therefore, peptide ligand residues X1 , X2, X5, X8 and X9 are grafted to the scaffold replacing residues Y1 , Y2, Y5, Y8 and Y9 with the corresponding outward facing residues peptide ligand residues X1 , X2, X5, X8 and X9, thereby creating an isomorphic replacement. The resultant grafted scaffold will have a grafted helix whose sequence would include the following residues: X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9 (Y10-30)

The resulting grafted helix preserves the native hydrogen bonding within the scaffold and at the same time preserves the noncovalent interactions required for specific binding of the peptide ligand to its target protein.

The“peptide ligand” may also contain more than one consecutive set of outward facing residues to graft into the scaffold, in which case the grafted scaffold may contain invariant scaffold residues between the grafted peptide residues (e, g“X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9”).

A helical peptide ligand may comprise all or part of the sequence C1X1X2C2X ₃ X4C ₃ X5X ₃ C4 , where Xi to Xe are independently any amino acid and Ci, C2, C ₃ and C4 are A, B, C and D, respectively, where A, B, C and D are invariant scaffold residues.

In some embodiments, a helical peptide ligand may be non-hydrophobic. For example, at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).

In some embodiments, a peptide ligand may be located at one or both termini of the grafted scaffold of the first to the fourteenth aspects. A peptide ligand located at the N or C terminus may comprise an a-helical structure and may comprise all or part of a half-repeat (i.e. all or part of a single a-helix) that stacks against the scaffold. The a-helix of the terminal peptide ligand makes stabilising interactions with the scaffold and is stable and folded. Only a few of the positions that structurally define an a-helix are required for the correct interfacial interaction with the scaffold. The residues in some of these positions are defined (Tyr (/) - lie i+4 ) - Tyr (i+7) - Leu (/-/- 11) for the N-terminal a-helix and Ala (/) - Leu ( i+4 ) - Ala/Val (i+7) for the C-terminal helix), but the remaining positions of the a-helix may be modified to form a helical peptide ligand.

A helical peptide ligand may be located at the N terminus of the scaffold of any one of the first to the fourteenth aspects. The N terminal peptide ligand may be helical and may comprise all or part of the sequence X _n -(X)i5-XiX2Xx, preferably all or part of the sequence X _r rXYxxXlxxYxxXLxx-XiX _2X x, where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, Xi is independently any amino acid, preferably D, and X2 is independently any amino acid, preferably P, and n is 0 or any number. In some embodiments, the Y, I, and/or L residues in the N terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).

A helical peptide ligand may be located at the C terminus of the scaffold of any one of the first to the fourteenth aspects. The C terminal peptide ligand may be helical and may comprise all or part of the sequence X _n -(X)i5-XiX2Xx, preferably all or part of the sequence Xi X ₂ XX-XXAXXXLXX[A or V]xxxxX-X _n , where X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, Xi is independently any amino acid, preferably D, and X2 is

independently any amino acid, preferably P, and n is 0 or any number. In some

embodiments, the A, L and/or V residues in the C terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).

The minimum length of the terminal peptide ligand is determined by the number of residues required to form a helix that binds to the target molecule. There is no intrinsic maximum length of the terminal peptide ligand and n may be any number.

In some embodiments, one or more positions in a peptide ligand may be diverse or randomised. A chimeric protein comprising a peptide ligand with one or more diverse or randomised residues may form a library as described below.

A chimeric protein of any one of the first to the fourteenth aspects may comprise peptide ligands in any arrangement or combination. For example, peptide ligands may be located in two or more loops of the scaffold; a loop and a helical region of the scaffold; or two or more helical regions of the scaffold. In some embodiments, a helical peptide ligand may be located at the N and/or C termini of the scaffold.

The minimum distance (MinD) between two peptide ligands within a chimeric protein may be conveniently determined from the crystal structure of the scaffold. Distance may be conveniently determined to an accuracy of +/- 7 A, which allows for variance of 2 residues (3.5 A x2). The maximum distance (MaxD) between two peptide ligands within a chimeric protein may be determined from MinD. When two peptide ligands are inserted into loops, the maximum distance between the ligands (MaxD) is MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2), wherein Xn is the number of amino acid residues in the loop. When two peptide ligands are inserted into helices, the maximum distance between the ligands (MaxD) is MinD. When one peptide ligand is inserted into a helix and the other is inserted into a loop, the maximum distance between the ligands (MaxD) is MinD + 1.75* Xn (loopl) wherein Xn is the number of amino acid residues in the loop. All MaxD have a propagated +/-5 A arising from the variance in MinD.

The distance between any two peptide ligands within a chimeric protein may be minimally at about 10 angstroms and maximally at about 30 A or more, 40 A or more, 50 A or more or 65 A or more, depending on the scaffold.

Generally, in CKS scaffolds of the first aspect, the peptide ligands are preferably 40 angstroms apart, they may be 35 angstroms, 30 angstroms, 20 angstroms, 15 angstroms but no less than 10 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a CKS scaffold may be 28 ± 7 A; MinD between a peptide ligand in the first loop and a peptide ligand in the third loop of a CKS scaffold may be 16 ± 7 A; MinD between a peptide ligand in the first loop and a peptide ligand in the helix of a CKS scaffold may be 19 ± 7 A; MinD between a peptide ligand in the second loop and a peptide ligand in the third loop of a CKS scaffold may be 27 ± 7 A; MinD between a peptide ligand in the second loop and a peptide ligand in the helix of a CKS scaffold may be 17 ± 7 A; and the MinD between a peptide ligand in the third loop and a peptide ligand in the fourth of a CKS scaffold may be 25 ± 7 A. The maximum distance (MaxD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a CKS scaffold may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2); MaxD between a peptide ligand in the first loop and a peptide ligand in the third loop of a CKS scaffold may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop3); MaxD between a peptide ligand in the first loop and a peptide ligand in the helix of a CKS scaffold may be MinD + 1.75* Xn (loopl); MaxD between a peptide ligand in the second loop and a peptide ligand in the third loop of a CKS scaffold may be MinD + 1.75* Xn (loop2) + 1.75 *Xn (loop3); MaxD between a peptide ligand in the second loop and a peptide ligand in the helix of a CKS scaffold may be MinD + 1.75* Xn (loop2); and the MaxD between a peptide ligand in the third loop and a peptide ligand in the fourth loop of a CKS scaffold may be MinD + 1.75* Xn (loop3) + 1.75* Xn (loop4).

Generally, in coiled-coil scaffolds of the second aspect, the peptide ligands are preferably 40 angstroms apart, they may be 35 angstroms, 30 angstroms, 20 angstroms, 15 angstroms but no less than 10 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a coiled-coil scaffold may be 10 ± 7 A. This may increase by about 3.6 A for every helical turn, or by 7.2 A every seven residues, depending on the location of the ligand in the helix. The maximum distance between a peptide ligand in the first loop and a peptide ligand in the helix of a coiled-coil scaffold may be is 52 ± 7 A. The minimum distance (MinD) between peptide ligands in the helices of a coiled-coil scaffold may be 17 ± 7 A. In some embodiments, the maximum distance (MaxD) between peptide ligands in the helices of a coiled-coil scaffold may be 50 ± 7 A (N terminal of one helix against C-terminal of the other helix). In other embodiments, the maximum distance may be greater than 50 ± 7 A, depending on the length of the coiled- coiled scaffold employed.

Generally in Affibody scaffolds of the third aspect, the peptide ligands are preferably 20 angstroms apart, they may be 18 angstroms, 15 angstroms, 12 angstroms, 11 angstrom, but no less than 10 angstroms apart. For example, the minimum distance (MinD) between peptide ligands in the helices of an Affibody scaffold may be 16 ± 7 A and the maximal distance (MaxD) may be 26 ± 7 A. The minimum distance (MinD) between a peptide ligand in the loop and a peptide ligand in the helix of an Affibody scaffold may be 16 ± 7 A and the maximal distance (MaxD) may be MaxD loop-helix = MinD + 1.75* Xn (loop).

Generally in Trefoil scaffolds of the fourth aspect, the peptide ligands are preferably 40 angstroms apart, they may be 35 angstroms, 32 angstroms, 30 angstroms, 28 angstroms but no less than 25 angstroms apart. For example, the minimum distance (MinD) between peptide ligands in opposing loops of a trefoild scaffold may be 24± 7 A and the maximal distance (MaxD) may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2).

Generally in PDZ scaffolds of the fifth aspect, the peptide ligands are preferably 40 angstroms apart, they may be 35 angstroms, 30 angstroms, 25 angstroms, 20 angstrom, but no less than 15 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a PDZ scaffold may be 30± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the fourth loop of a PDZ scaffold may be 31± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop4). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the third loop of a PDZ scaffold may be 29± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2) + 1.75 *Xn (loop3). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the fourth loop of a PDZ scaffold may be 27± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3) + 1.75 *Xn (loop4). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a PDZ scaffold may be 19± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the helix of a PDZ scaffold may be 18± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the helix of a PDZ scaffold may be 16± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3). The minimum distance (MinD) between a peptide ligand in the fourth loop and a peptide ligand in the helix of a PDZ scaffold may be 17± 7 A and the maximum distance may be MinD + 1.75* Xn (loop4). Preferably, peptide ligands are not grafted in the first and third loops of a PDZ scaffold or the second and fourth loops of a PDZ scaffold.

Generally in Ubiquitin scaffolds of the sixth aspect, the peptide ligands are preferably 25 angstroms apart, they may be 22 angstroms, 20 angstroms, 18 angstroms, 16 angstroms but no less than 15 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a Ubiquitin scaffold may be 23± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the third loop of a Ubiquitin scaffold may be 23± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop3). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a Ubiquitin scaffold may be 20± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the helix of a ubiquitin scaffold may be 15± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the helix of a Ubiquitin scaffold may be 21± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3). Preferably, the two peptide ligands are not grafted into the second and third loops of a Ubiquitin scaffold.

Generally in GB1 scaffolds of the seventh aspect, the peptide ligands are preferably 20 angstroms apart, they may be 21 angstroms, 22 angstroms, 24 angstroms, but no less than 19 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a GB1 scaffold may be 29± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the fourth loop of a GB1 scaffold may be 26± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop4). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the third loop of a GB1 scaffold may be 26± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2) + 1.75 *Xn (loop3). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the fourth loop of a GB1 scaffold may be 22± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3) + 1.75 *Xn (loop4). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a GB1 scaffold may be 26± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the helix of a GB1 scaffold may be 20± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the helix of a GB1 scaffold may be 16± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3). The minimum distance (MinD) between a peptide ligand in the fourth loop and a peptide ligand in the helix of a GB1 scaffold may be 10± 7 A and the maximum distance may be MinD + 1.75* Xn (loop4).

Generally, in VWV scaffolds of the eighth aspect, the distance between two peptide ligands in the loops or helical region of the VWV scaffold may be 10A or more, or 15A or more. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a VWV scaffold may be 20± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2).

Generally, in Fibritin scaffolds of the ninth aspect, the distance between two peptide ligands in the coiled-coil domain and disordered regions of the Fibritin scaffold may be 30A or more, 40A or more, 50A or more or 65A or more. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a Fibritin scaffold may be 70± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a Fibritin scaffold may be 39± 7 A and the maximum distance may be 46± 7 A. The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the helix of a Fibritin scaffold may be 26± 7 A and the maximum distance may be 102± 7 A.

Generally in aPP scaffolds of the tenth aspect, the peptide ligands are preferably 40 angstroms apart, they may be 35 angstroms, 30 angstroms, 25 angstroms, but no less than 20 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of an aPP scaffold may be 19± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl).

Generally, in fibronectin scaffolds of the eleventh aspect, the distance between two peptide ligands may be 30A or more, 40A or more, 50A or more or 65A or more. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a fibronectin scaffold may be 40± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the fourth loop of a fibronectin scaffold may be 38± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop4). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the third loop of a fibronectin scaffold may be 34± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2) + 1.75 *Xn (loop3). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the fourth loop of a fibronectin scaffold may be 30± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3) + 1.75 *Xn (loop4). Preferably, the two peptide ligands are not grafted into the first and third loops or the second and fourth loops of a fibronectin scaffold.

Generally in Zn finger scaffolds of the twelfth aspect, the peptide ligands are preferably 20 angstroms apart, they may be 19 angstroms, 18 angstroms, or 16 angstroms, but no less than 15 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of a ZNF scaffold may be 21± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the helix of a ZNF scaffold may be 19± 7 A and the maximum distance (MaxD) may be MinD + 1.75* Xn (loopl). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the helix of a ZNF scaffold may be 12± 7 A and the maximum distance may be MinD + 1.75* Xn (loop2).

Generally in SH3 scaffolds of the thirteenth aspect, the peptide ligands are preferably 30 angstroms apart, they may be 25 angstroms, 20 angstroms, or 17 angstroms, but no less than 15 angstroms apart. For example, the minimum distance (MinD) between a peptide ligand in the first loop and a peptide ligand in the second loop of an SH3 scaffold may be 21 ± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). The minimum distance (MinD) between a peptide ligand in the second loop and a peptide ligand in the third loop of an SH3 scaffold may be 26± 7 A and the maximum distance (MaxD) may be MinD + 1.75* Xn (loop2) + 1.75 *Xn (loop3). The minimum distance (MinD) between a peptide ligand in the third loop and a peptide ligand in the fourth loop of an SH3 scaffold may be 22± 7 A and the maximum distance may be MinD + 1.75* Xn (loop3) + 1.75 *Xn (loop4).

Generally in CK scaffolds of the fourteenth aspect, the distance between two peptide ligands in the loops may be 10A or more, or 15A or more. For example, the minimum distance (MinD) between a peptide ligand in a first loop and a peptide ligand in a second loop of a CK scaffold may be 8± 7 A and the maximum distance may be MinD + 1.75* Xn (loopl) + 1.75 *Xn (loop2). A person of skill in art can use 3D software such as Chimera or Pymol to determine the minimum distances between positions for ideal positioning in three-dimensional orientation.

The location of the peptide ligands within a chimeric protein may be determined by rational design, for example using modelling to identify the optimal arrangement for the presentation of two target molecules to each other (e.g. for substrate presentation to an E3 ubiquitin ligase); and/or by screening for example using populations of chimeric protein with different arrangements of peptide ligands to identify the arrangement which confers the optimal interaction of target molecules.

Target Molecules

Suitable target molecules for chimeric proteins of any one of the first to the fourteenth aspects include biological macromolecules, such as proteins. The target molecule may be a receptor, enzyme, antigen, oligosaccharide, oligonucleotide, integral membrane protein, transcription factor, transcriptional regulator, G protein-coupled receptor (GPCR) or any other target of interest. Targets that are difficult to drug with small molecules, such as PPIs, proteins that accumulate in neurodegenerative diseases and proteins overexpressed in disease conditions, such as cancer, may be particularly suitable target molecules. Target molecules may include a-synuclein; b-amyloid; tau; superoxide dismutase; huntingtin; b- catenin; KRAS; Bcl-2, Bcl-xL; components of superenhancers and other types of transcriptional regulators, such as N-Myc, c-Myc, Notch, , EWS-FLI 1 (Ewing’s sarcoma- friend leukemia integration 1), CtBP, Bcl9, Jun, Fos, TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1) Sox2 ((sex determining region Y)-box 2), BFL1 ; tankyrases; phosphatases such as PP2A and PP1c; epigenetic writers, readers and erasers, such as histone deacetylases and histone methyltransferases; BRD4 and other bromodomain proteins; and kinases, such as PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), Aurora A, Cdk2, GSK3, CK2-alpha and RTK; WDR5, EED, MCL1 , GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

In some embodiments, a chimeric protein may neutralise a biological activity of the target molecule, for example by inhibiting or antagonising its activity or binding to another molecule or by tagging it for ubiquitination and proteasomal degradation or for degradation via autophagy and lysosomal pathways. In other embodiments, a chimeric protein may activate a biological activity of the target molecule.

In some embodiments, the target molecule may be b-catenin. Suitable peptide ligands that specifically bind to b-catenin are well-known in the art and include b-catenin- peptide ligands derived from axin (e.g. GAYPEYI LDI H VYRVQLEL and variants thereof), Bcl-9 (e.g.

SQEQLEHRYRSLITLYDIQLML and variants thereof), TCF7L2 (e.g.

QELGDNDELMHFSYESTQD and variants thereof), I CAT (e.g. YAYQRAIVEYMLRLMS and variants thereof), LRH-1 (e.g. YEQAIAAYLDALMC and variants thereof), A PC (e.g.

SCSEELEALEALELDE and variants thereof), a-catenin (e.g.

RSKKAHVLAASVEQATQNFLEKGEQIAKESQ or RTLTVERLLEPLVTQVTTLV and variants thereof), A PC membrane recruitment protein (e.g.

RREQLEAQEARAREAHAREAHAREAYTREAYGREAYAREAHTWEAHGREARTREAQA

and variants thereof, SOX (e.g. DxxEFDQYL and variants thereof) and kindlin 2 (e.g.

QALLDKAKINQ GWLDSSRSLMEQDKENEALLRF and variants thereof).

In some embodiments, the target molecule may be KRAS. Suitable peptide ligands that specifically bind to KRAS are well-known in the art and include a KRAS-peptide ligand from SOS-1 (e.g. FEGIALTNYLKALEG and variants thereof), synthetic peptide 225-1 (e.g SIEDLHEYWARLWNYLYVA and variants thereof), synthetic peptide 225-15a (e.g

QASLEELHEYWARLWNYRVA and variants thereof), synthetic peptide 225-15b (e.g NASI KQLHAYWQRLYAYLAAVA and variants thereof), phage-display library peptide KR- pep3 (e.g. CMWWREICPVWW and variants thereof), Raf-S (e.g. FARKTFLKLAF and variants thereof), NF1 (ARRFFLDIAD and variants thereof), Rasin peptide 2 (FRWPxxRLxx and variants thereof), Rasin peptide 1 (txVFxhxp and variants thereof), NF1 monobody peptide (74-84) (YGHGQVYYY and variants thereof), farnesyl transferase 1

(DAYECLDASRPW and variants thereof), farnesyl transferase 2 (ENPKQN and variants thereof), farnesyl transferase 3 (KSRDFYH and variants thereof) and KRAS-peptide ligands identified by phage display (see for example Sakamoto et al. Biochem. Biophys. Res.

Comm. (2017) 484 605-611).

In some embodiments, the target molecule may be tankyrase. Suitable peptide ligands that specifically bind to tankyrase are well-known in the art and include tankyrase peptide ligands from Axin (e.g. REAGDGEE and HLQREAGDGEEFRS or variants thereof).

In some embodiments, the target molecule may be EWS-FLI1. Suitable peptide ligands that specifically bind to EWS-FLI1 are well-known in the art and include the ESAP1 peptide TMRGKKKRTRAN and variants thereof. Other suitable sequences may be identified by phage display (see for example Erkizan et al. Cell Cycle (2011) 10, 3397-408).

In some embodiments, the target molecule may be Aurora-A. Suitable peptide ligands that specifically bind to Aurora-A are well-known in the art and include Aurora-A binding sequences from TPX2, such as SYSYDAPSDFINFSS (Bayliss et al. Mol. Cell (2003) 12, 851-62) and Aurora-A binding sequences from N-myc, such as N-myc residues 19-47 or 61- 89 (see for example Richards et al. PNAS (2016) 113, 13726-31).

In some embodiments, the target molecule may be N-Myc or C-Myc. Suitable peptide ligands that specifically bind to N-myc or C-myc are well-known in the art and include binding sequences from Aurora-A (see for example Richards et al. PNAS (2016) 113, 13726-31)

(e.g. AGVEHQLRREVEIQSH, WSVHAPSSRRTTpLAGTLDYLPPEMI and TYQETY and variants thereof), Omomyc (e.g. QAEEQKLSEEDLLR KRREQLKHKLEQLRNSCA and variants thereof), H1 F8A (e.g. NELKRSFAALRDQI or variants thereof), H1 F8A S6A (e.g. NELKRAFAALRDQI or variants thereof), MIP (e.g.

IREKNHYHRQEVDDLRRQNALLEQQVRAL or variants thereof), PIN1 (e.g. FNHITNASQWE or variants thereof), PIN2 (e.g. GDLGAFSRGQM or variants thereof), 9E10 paratope (e.g. RSEFYYYGNTYYYSAMD or variants thereof), BIN1 (e.g. QHDYTATDE, QNPEEQDEGW or EKCRGVFPENF or variants thereof).

In some embodiments, the target molecule may be WDR5 (WD repeat-containing protein 5). Suitable peptide ligands that specifically bind to WDR5 are well-known in the art and include the WDR5-interacting motif (WIN) of MLL1 (mixed lineage leukemia protein 1) (see for example Song & Kingston J. Biol. Chem. (2008) 283, 35258-64; Patel et al. J. Biol. Chem. (2008) 283, 32158-61), e.g. EPPLNPHGSARAEVHLRKS and variants thereof.

In some embodiments, the target molecule may be BRD4 or a Bromodomain protein.

Suitable peptide ligands that specifically bind to BRD4 are well-known in the art and include sequences derived from histone protein ligands and binding sequences from JMJD6 (e.g. KWTLERLKRKYRN and variants thereof) and murine leukemia virus integrase (e.g.

TWRVQRSQNPLKIRLTR and variants thereof).

In some embodiments, the target molecule may be a HDAC (histone deacetylase). Suitable peptide ligands that specifically bind to HDAC are well-known in the art and include binding sequences derived from SMRT and other proteins that recruit HDACs to specific

transcriptional regulatory complexes or binding sequences derived from histone proteins (see for example Watson et al. Nat. Comm. (2016) 7, 11262; Dowling et al. Biochem. (2008) 47, 13554-63).

In some embodiments, the target molecule may be Notch. Suitable peptide ligands that specifically bind to Notch are well-known in the art and include binding sequences from the N-terminus of MAML1 (mastermind like protein 1), e.g. SAVMERLRRRIELCRRHHST and variants thereof (see for example Moellering et al. Nature (2009) 462, 182-8).

In some embodiments, the target molecule may be a Cdk (cyclin-dependent kinase).

Suitable peptide ligands that specifically bind to Cdks are well-known in the art and include substrate- based peptides, for example, Cdk2 sequences derived from cyclin A, such as TYTKKQVLRMEHLVLKVLTFDL and variants thereof (see for example Gondeau et al. J.

Biol. Chem. (2005) 280, 13793-800; Mendoza et al. Cancer Res. (2003) 63, 1020-4).

In some embodiments, the target molecule may be PLK1 (polo-like kinase 1). Suitable peptide ligands that specifically bind to PLK1 are well-known in the art and include optimised substrate-derived sequences that bind to the substrate-binding PBD (polo- box domain), such as MAGPMQSEPLMGAKK and variants thereof.

In some embodiments, the target molecule may be Tau. Suitable peptide ligands that specifically bind to Tau are well-known in the art and include tau-binding sequences derived from alpha- and beta-tubulin, such as KDYEEVGVDSVE and YQQYQDATADEQG and variants thereof (see for example Maccioni et al. EM BO J. (1988) 7, 1957-63; Rivas et al. PNAS (1988) 85, 6092-6).

In some embodiments, the target molecule may be BCR-ABL. Suitable peptide ligands that specifically bind to BCR-ABL are well-known in the art and include optimized substrate- derived sequences, such as EAIYAAPFAKKK and variants thereof.

In some embodiments, the target molecule may be PP2A (protein phosphatase 2A). Suitable peptide ligands that specifically bind to PP2A are well-known in the art and include sequences that bind the B56 regulatory subunit, such as LQTIQEEE and variants thereof (see for example Hetz et al. Mol. Cell (2016), 63 686-95).

In some embodiments, the target molecule may be EED (Embryonic ectoderm

development). Suitable peptide ligands that specifically bind to EED are well-known in the art and include helical binding sequences from co-factor EZH2 (enhancer of zeste homolog 2), such as FSSNRQKILERTEILNQEWKQRRIQPV and variants thereof (see for example Kim et al. Nat. Chem. Biol. (2013) 9, 643-50.)

In some embodiments, the target molecule may be MCL-1 (induced myeloid leukemia cell differentiation protein). Suitable peptide ligands that specifically bind to MCL-1 are well- known in the art and include sequences from BCL2, e.g. KALETLRRVGDGVQRNHETAF and variants thereof (see for example Stewart et al. Nat. Chem. Biol. (2010) 6, 595-601).

In some embodiments, the target molecule may be RAS. Suitable RAS peptide ligands are well-known in the art and include RAS-binding peptides identified by phage display, such as RRRRCPLYISYDPVCRRRR and variants thereof (see for example Sakamoto et al. BBRC (2017) 484, 605-11).

In some embodiments, the target molecule may be GSK3 (glycogen synthase kinase 3). Suitable GSK3 peptide ligands are well-known in the art and include substrate-competitive binding sequences such as KEAPPAPPQDmcH P, LSRRPDYR, RREGGMSRPADVDG, and YRRAAVPPSPSLSRHSSPSQDEDEEE and variants thereof (see for example llouz et al. J. Biol. Chem. 281 (2006), 30621-30630. Plotkin et al. J. Pharmacol. Exp. Ther. (2003) 305, 974-980).

In some embodiments, the target molecule may be CtBP (C-terminal binding protein).

Suitable CtBP peptide ligands are well-known in the art and include sequences identified from a cyclic peptide library screen, such as SGWTWRMY and variants thereof (see for example Birts et al. Chem. Sci. (2013) 4, 3046-57).

Examples of suitable peptide ligands for target molecules that may be used in a chimeric protein of any one of the first to the fourteenth aspects are shown in Tables 1 , 2 and 6 or variants thereof. Other suitable peptide ligands are highlighted (bold) in Tables 4, 5, 12, 13, 16, 18, 20, 23, 25, 26, 29, 30, 33, 38, 41 , 45, 48, and 51 (SEQ ID NOs 660-840).

In some preferred embodiments, a chimeric protein of any one of the first to the fourteenth aspects may comprise a peptide ligand for an E3 ubiquitin ligase. Examples of suitable E3 ubiquitin ligases include MDM2, SCF ^Sk P ² , BTB-CUL3-RBX1 , APC/C, SIAH, CHIP, Cul4- DDB1 , SCF-family, b-TrCP, Fbw7 and Fbx4.

Suitable peptide ligands for E3 ubiquitin ligases (degrons) are well known in the art and may be 3 to 30 amino acids, 3 to 25 amino acids, 3 to 20 amino acids, or any length

therebetween. For example, a suitable peptide ligand for MDM2 may include a peptide ligand from p53 (e.g. FAAYWNLLSAYG, RFMDYWEGL orTSFAEYWALLAENL) or a variant thereof. A suitable peptide ligand for SCF ^Skp2 may include a peptide ligand from p27 (e.g. AGSNEQEPKKRS) and variants thereof. A suitable peptide ligand for Keap1-Cul3 may include a peptide ligand from Nrf2 (e.g. DPETGEL) or a variant thereof. A suitable peptide ligand for SPOP-Cul3 may be include a peptide ligand from Puc (e.g. LACDEVTSTTSSSTA) or a variant thereof. A suitable peptide ligand for A PC/C may include the degrons termed ABBA (e.g. SLSSAFHVFEDGNKEN), ABOX (QRVL), KEN (e.g. SEDKENVPP), or DBOX (e.g. PRLPLGDVSNN) or a variant thereof. In some instances, a combination of these degrons may be used (mimicking the bipartite or tripartite degrons found in some natural substrates). A suitable peptide ligand for SIAH may include a peptide ligand from PHYL (e.g. LRPVAMVRPTV) or a variant thereof. A suitable peptide ligand for SCF ^Fbw7 may include a peptide ligand from cyclin-D3 (e.g. PEQTSEPTDVAI or SLIPEPDR) or a variant thereof. A suitable peptide ligand for SCF ^Fbw8 may include a peptide ligand from cyclin-D3 (e.g.

EPPLEP) or a variant thereof.

A suitable peptide ligand for Cul4-DDB1-Cdt2 may include a peptide ligand from PIP, such as QRRMTDFYARRR or a variant thereof. A suitable peptide ligand for CHIP (carboxyl terminus of Hsc70- interacting protein) may include peptide sequences such as ASRMEEVD (from Hsp90 C-terminus) and GPTIEEVD (from Hsp70 C-terminus) or a variant thereof. A suitable peptide ligand for beta-TrCP may include a degron sequence motif (including phosphomimetic amino acids), such as DDGYFD or a variant thereof. A suitable peptide ligand for Fbx4 may include sequences derived from TRF1 , such as

MPIFWKAHRMSKMGTG or a variant thereof (see for example Lee et al. Chembiochem (2013) 14, 445-451). A suitable peptide ligand for FBw7 may include degron sequence motifs (including phosphomimetic amino acids), such as LPSGLLEPPQD. A suitable peptide ligand for DDB1-Cul4 may include sequences derived from HBx (hepatitis B virus X protein), such as ILPAVLHLRTVYG and variants thereof and similar proteins from other viruses, such as TVAYFTLQQVYG and NFVAWHALRQVYG and variants thereof and from DCAFs (DDB1-CUL4-associated factors) including helical motifs such as ILPKVLHKRTLGL, NFVSWHANRQLGM, NTVEYFTSQQVTG, and NITRDLIRRQIKE (see for example Li et al. Nat. Struct. Mol. Biol. (2010) 17, 105-111). A suitable peptide ligand for DDB1-Cul5 may include sequences derived from DCAF9, such as NITADLILRQVYG and variants thereof.

Examples of suitable peptide ligands for E3 ubiquitin ligases that may be used in a chimeric protein of any one of the first to the fourteenth aspects include the sequences shown in Table 2 and variants thereof. Other suitable peptide ligands are highlighted (bold, dashed underline) in Tables 4, 5, 12, 13, 16,18, 20, 23, 25, 26, 29, 30, 33, 38, 41 , 45, 48, and 51 (SEQ ID NOs 660-840).

A suitable peptide ligand for an E3 ligase also may be selected from a library, for example using phage or ribosome display, or identified or designed using rational approaches or computational design, for example using the crystal structure of a complex or an interaction. In some embodiments, peptide ligands may be identified in an amino acid sequence using standard sequence analysis tools (e.g. Davey et al Nucleic Acids Res. 2011 Jul 1 ; 39 (Web Server issue): W56-W60).

A chimeric protein comprising a peptide ligand for an E3 ubiquitin ligase may also comprise a peptide ligand for a target molecule. Binding of the chimeric protein to both the target molecule and the E3 ubiquitin ligase may cause the target molecule to be ubiquitinated by the E3 ubiquitin ligase. Ubiquitinated target molecules are then degraded by the

proteasome. This allows the specific targeting of molecules for proteolysis by the chimeric protein. The ubiquitination and subsequent degradation of a target protein has been shown for hetero-bifunctional small molecules (PROTACs; proteolysis targeting chimeras) that bind the target protein and a ubiquitin ligase simultaneously (see for example Bondeson et al.

Nat. Chem. Biol. 2015; Deshaies 2015; Lu et al. 2015).

In some embodiments, the chimeric protein may lack lysine residues, so that it avoids ubiquitination by the E3 ubiquitin ligase. Examples of chimeric proteins that bind E3 ubiquitin ligase and a target molecule are shown in Tables 4, 5, 12, 13, 16,18, 20, 23, 25,

26, 29, 30, 33, 38, 41 , 45, 48, 51 , 55 and 56 (SEQ ID NOs 660-840).

In some preferred embodiments, a chimeric protein as described herein may comprise an amino acid shown in any one of Tables 4, 12, 16, 20, 25, 29, 33, 38, 41 , 45, 48, 51 , 55, 56 or a variant thereof (SEQ ID NOs 660-840).

In other preferred embodiments, a chimeric protein of any one of the first to the fourteenth aspects may comprise a peptide ligand that binds to a component of a target-selective autophagy pathway, such as chaperone-mediated autophagy (CMA). The chimeric protein and target molecules bound thereto are thus recognised by the autophagy pathway and the target molecules are subsequently degraded. Suitable components of the CMA pathway include heat shock cognate protein of 70 kDa (hsc70, HSPA8, Gene ID: 3312). Suitable peptide ligands are well known in the art (Dice J.F. (1990). Trends Biochem. Sci. 15, 305- 309) and include W/F/Y]xx[L/l/V], Lys-Phe-Glu-Arg-Gln (KFERQ), QRFFE and variants thereof, such as CMA_Q and CMA_K, as described herein. These domains have been demonstrated to be capable of targeting heterologous proteins to the autophagy pathway (Fan, X.et al; (2014) Nature Neuroscience 17, 471-480).

In other preferred embodiments, a chimeric protein of any one of the first to the fourteenth aspects may comprise a peptide ligand that binds to a component of a lysosomal degradation pathway, such as the ESCRT (endosomal sorting complexes required for transport) pathway. The chimeric protein and target molecules bound thereto are thus recognised by the lysosomal degradation pathway and the target molecules are

subsequently degraded. This may be useful for example when the target molecule is a membrane protein. Suitable components of the ESCRT pathway include ALIX and Adapter Protein-1 , -2 or -3. Suitable peptide ligands are well known in the art and include ALIX binding sequences derived from Gag, such as LYPxxxL and variants thereof, and AP binding sequences, such as ExxxLL, YxxL, SREKPYKEVTEDLLHLNSLF and

AAGAYDPARKLLEQYAKK and variants thereof or the lysosomal targeting sequence KFERQQKILDQRFFE and variants thereof.

Examples of suitable peptide ligands for autophagy or for other degradation pathways that may be used in a chimeric protein of any one of the first to the fourteenth aspects include the sequences shown in Table 2 and variants thereof.

In other preferred embodiments, degradation of the target molecule may be conditional upon phosphorylation of one of the peptide ligands of the chimeric protein. Peptide ligands whose activity is dependent on their phosphorylation status e.g. they are functional when phosphorylated and non-functional when not phosphorylated may be termed conditional peptide ligands. A chimeric protein of any one of the first to the fourteenth aspects may comprise one or more conditional peptide ligands. For example, a conditional peptide ligand may bind to a target molecule, such as an SH2, PTB (phosphotyrosine-binding) or WW domain of a disease-associated target protein, when phosphorylated, but not bind to the target molecule when not phosphorylated. A conditional peptide ligand (“degron”) may bind to the SH2, PTB or WW domain of an E3 ligase or another member of a cellular degradation pathway when phosphorylated, but not bind to the E3 ligase or other member when not phosphorylated. In some preferred embodiments, the kinase that phosphorylates peptide ligand is upregulated in the disease being targeted. This allows the selective degradation of the target molecule in disease cells.

Additional Functional Domains and Labels

In addition to scaffolds and peptide ligands, a chimeric protein of any one of the first to the fourteenth aspects may further comprise one or more additional domains which confer additional functionality, such as targeting domains, intracellular transport domains, stabilising domains or oligomerisation domains. Additional domains may for example be located at the N or C terminus of the chimeric protein or in a loop between repeats. A targeting domain may be useful in targeting the chimeric protein to a particular destination in vivo, such as a target tissue, cell, membrane or intracellular organelle. Suitable targeting domains include chimeric antigen receptors (CARs).

An intracellular transport domain may facilitate the passage of the chimeric protein through the cell membrane into cells, for example to bind intracellular target molecules. Suitable intracellular transport domains are well known in the art (see for example Bechara et al FEBS Letters 587 1 (2013) 1693-1702) and include cell-penetrating peptides (CPFs), such as Antennapedia (43-58), Tat (48-60), Cadherin (615-632) and poly-Arg.

Other suitable intracellular transport domains include pepducins. Pepducins are synthetic lipidated peptides comprising amino acid sequences from specific intracellular loops of GPCRs and other membrane proteins that are known to move across the plasma

membrane, resulting in the delivery of the peptidic component being delivered to the cytoplasm. A pepducin may be conjugated to the grafted CKS scaffold using any convenient technique, for example, native chemical ligation, click chemistry or cysteine chemistry If expression of the GPCR or other membrane protein is up regulated in the disease being targeted, then delivery of the chimeric protein would be selectively targeted to disease cells.

Other suitable intracellular transport domains include receptor ligands (e.g. GLP1 (glucagon like peptide 1)) that bind the extracellular domains of a receptor (e.g. GLP1 R) and activate it, leading to peptide-bound receptor internalisation These receptor ligands have been shown to be capable of delivering conjugated cargo macromolecules (e.g. nucleic acids) into cells.

A receptor ligand may be conjugated to the grafted CKS scaffold using any convenient technique, for example native chemical ligation or cysteine chemistry. If expression of the receptor is upregulated in the disease being targeted, then delivery of the chimeric protein would be selectively targeted to disease cells.

A stabilising domain may increase the half-life of the chimeric protein in vivo. Suitable stabilising domains are well known in the art and include Fc domains, serum albumin, unstructured peptides such as XTEN ⁹⁸ or PAS" and polyethylene glycol (PEG).

An oligomerisation domain may facilitate the formation of multi-protein complexes, for example to increase avidity against multi-valent targets. Suitable oligomerisation domains include the‘foldon’ domain, the natural trimerisation domain of T4 Fibritin (Meier et al., J.

Mol. Biol. (2004) 344(4): 1051-69). In addition to scaffolds, peptide ligands and optionally one or more additional domains, a chimeric protein of any one of the first to the fourteenth aspects may further comprise a cytotoxic or therapeutic agent and/or detectable label.

Suitable cytotoxic agents include, for example, chemotherapeutic agents, such as methotrexate, a uri statin adriamicin, doxorubicin, melphalan, mitomycin C, ozogamicin, chlorambucil, maytansine, emtansine, daunorubicin or other intercalating agents,

enzymatically active toxins of bacterial, fungal, plant, or animal origin, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, a-amanitin, alpha-sarcin, Aleurites fordii proteins, tubulysins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, pyrrolobenzodiazepines, and the tricothecenes and fragments of any of these. Suitable cytotoxic agents may also include radioisotopes. A variety of radionuclides are available for the production of radioconjugated chimeric proteins including, but not limited to, ⁹⁰ Y, ¹²⁵ l, ¹³¹ l, ¹²³ l, ¹¹¹ In, ¹³¹ ln, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho,

¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re and ²¹² Bi. Conjugates of a chimeric protein and one or more small anti cancer molecules, for example toxins, such as a calicheamicin, maytansinoids, a

trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, may also be used.

Suitable therapeutic agents may include cytokines (e.g. IL2, IL12 and TNF), chemokines, pro-coagulant factors (e.g. tissue factor), enzymes, liposomes, and immune response factors.

A detectable label may be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. Detectable labels may be attached to chimeric protein using conventional chemistry known in the art.

There are numerous methods by which the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents. The label can also be bound to another specific binding member that binds the chimeric protein, or to a support.

In some embodiments, 1 , 2, 3, 4, 5 or more, preferably all of the amino acids in a chimeric protein of any one of the first to the fourteenth aspects may be D-amino acids. This may be useful for example in minimising immunogenicity and protease-sensitivity. The production of proteins comprising D amino acids is well-known in the art (see for example Garton et al (2018) Proc Natl Acad Sci USA 115 1505-1510).

In some embodiments, the grafted scaffold of a chimeric protein of any one of the first to the fourteenth aspects may be cyclized i.e. the N and C termini of the scaffold may be covalently linked. This may be useful for example in improving stability and protease-resistance. The N- and C-termini of the scaffold may be covalently linked using any convenient technique, for example, native chemical ligation or disulphide bonds. Suitable cyclization techniques are known in the art (see for example Deechongkit & Kelly JACS (2002) 124 4980-4986; Patel et al (2013) Protein Eng Des Sel 26 307-315; Camarero et al. J Mol Biol (2001) 308 1045- 1062; Schuman et al. Frontiers Chem (2015) 3).

In some embodiments, a chimeric protein may be configured for display on a particle or molecular complex, such as a cell, ribosome or phage, for example for screening and selection. A suitable chimeric protein may further comprise a display moiety, such as phage coat protein, to facilitate display on a particle or molecular complex. The phage coat protein may be fused or covalently linked to the chimeric protein.

Production of Chimeric Proteins

Chimeric proteins as described herein may be produced by recombinant means. For example, a method of producing a chimeric protein of any one of the first to the fourteenth aspects may comprise expressing a nucleic acid encoding the chimeric protein. A nucleic acid may be expressed in a host cell and the expressed chimeric protein may then be isolated and/or purified from the cell culture.

In some embodiments of any one of the first to the fourteenth aspects, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a scaffold to produce a chimeric nucleic acid encoding a chimeric protein of any one of the first to the fourteenth aspects comprising a peptide ligand located in the scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

In some embodiments of the first aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a CKS scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located in one of the first loop, second loop, third loop or helical region of the CKS scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold and a second peptide ligand located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a CKS scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a CKS scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold.; and

expressing the nucleic acid to produce said protein. In some embodiments of the second aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a coiled-coil scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 16 to 55 or between residues 59 to 83 of any one of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a coiled-coil scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a coiled-coil scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the third aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an Affibody scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID NOs: 54, 56 and 58 to 78) of the Affibody scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding an Affibody scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 54, 56 and 58 to 78) of the Affibody scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding an Affibody scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 54, 56 and 58 to 78) of the Affibody scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein.

In some embodiments of the fourth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Trefoil scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a Trefoil scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a Trefoil scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues

21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the

Trefoil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein. In some embodiments of the fifth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a PDZ scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a PDZ scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the SEQ ID NO:79) of the PDZ scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a PDZ scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO:79) of the PDZ scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the sixth aspect, a method may comprise; inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Ubiquitin scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305) of the Ubiquitin scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a Ubiquitin scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299,

303 and SEQ ID NO: 305) of the Ubiquitin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a Ubiquitin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305) of the Ubiquitin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the seventh aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a GB1 scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305) of the GB1 scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a GB1 scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305) of the of the GB1 scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a GB1 scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and SEQ ID NO: 305) of the GB1 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the eighth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a VWV scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located in the first loop or second loop of the VWV scaffold or the helical region, if present; and, expressing said chimeric nucleic acid to produce the chimeric protein.

Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the e first loop or second loop of the VWV scaffold or the helical region, if present; and a second peptide ligand located at another of the first loop or second loop of the V V scaffold or the helical region, if present.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a VWV scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said first and second peptide ligands are located in (a) the first and second loops,

(b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a VWV scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the ninth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Fibritin scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located in the coiled-coil subdomain or disordered region of the Fibritin scaffold of the chimeric protein; and, expressing said chimeric nucleic acid to produce the chimeric protein.

Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and a second peptide ligand located at another of the coiled-coil subdomain and disordered region of the Fibritin scaffold.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a Fibritin scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a Fibritin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold; and

expressing the nucleic acid to produce said protein.

In some embodiments of the tenth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an aPP scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located in the loop or the helical region of the aPP scaffold of the chimeric protein; and, expressing said chimeric nucleic acid to produce the chimeric protein.

Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the loop and helical region of the aPP scaffold and a second peptide ligand located at another of the loop and helical region of the aPP scaffold.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding an aPP scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located at one of the loop and helical region of the aPP scaffold and the second peptide ligand is located at the other of the loop and helical region of the aPP scaffold; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding an aPP scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein the first peptide ligand is located at one of the loop and helical region of the aPP scaffold and the second peptide ligand is located at the other of the loop and helical region of the aPP scaffold; and

expressing the nucleic acid to produce said protein.

In some embodiments of the eleventh aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a fibronectin scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at one of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein. Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the first, second, third and fourth loops of the fibronectin scaffold and a second peptide ligand located at another of the first, second, third and fourth loops of the fibronectin scaffold.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a fibronectin scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a fibronectin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold; and

expressing the nucleic acid to produce said protein.

In some embodiments of the twelfth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Zn finger scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein. Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the first loop, second loop and helical region of the Zn finger scaffold and a second peptide ligand located at another of the first loop, second loop and helical region of the Zn finger scaffold.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a Zn finger scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding a Zn finger scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the thirteenth aspect, a method may comprise;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an SH3 scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at SH3 of the SH3 scaffold of the chimeric protein; and, expressing said chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding an SH3 scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at SH3 of the SH3 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

providing a nucleic acid encoding an SH3 scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located at SH3 of the SH3 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

In some embodiments of the fourteenth aspect, a method may comprise;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a cystine knot scaffold to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein; and,

expressing said chimeric nucleic acid to produce the chimeric protein.

Preferably, a third nucleic acid encoding a second peptide ligand is inserted into the second nucleic acid to produce a chimeric nucleic acid encoding a chimeric protein comprising a first peptide ligand located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and a second peptide ligand located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold. Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

providing a nucleic acid encoding a cystine knot scaffold, and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein each peptide ligand is located in one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

An isolated nucleic acid encoding a chimeric protein of any one of the first to the fourteenth aspects is provided as an aspect of the invention. The nucleic acid may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.

Many techniques and protocols that are suitable for the expression of recombinant chimeric protein in cell culture and their subsequent isolation and purification are known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (Mar 1997) Humana Press Inc). Wthin this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A laboratory Manual (Sambrook, et al. 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 85 edited by D. Goeddel, 1991 Academic Press, San Diego, A), "Guide to Protein Purification" Methods in Enzymology (MP. Deutshcer, ed. (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, Ed. (R.1. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-1 28, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 98 Catalog (Ambion, Austin, TX).

A host cell comprising a nucleic acid encoding a chimeric protein of any one of the first to the fourteenth aspects or vector containing such a nucleic acid is also provided as an aspect of the invention. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of proteins in prokaryotic cells is well established in the art. A common bacterial host is E. coli. A chimeric protein may also be produced by expression in eukaryotic cells in culture.

Mammalian cell lines available in the art for expression of a chimeric protein include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells) and many others.

In other embodiments, chimeric proteins of any one of the first to the fourteenth aspects may be produced by synthetic means. Suitable methods for the chemical synthesis of peptidyl molecules are well known in the art. This may be useful for example in allowing the incorporation of unnatural amino acids and/or additional domains or chemical groups which confer additional functionality, such as molecular recognition and enhanced proteolytic stability.

Libraries of Chimeric Proteins

Chimeric proteins of any one of the first to the fourteenth aspects may be used to produce libraries. A suitable library may be screened in order to identify and isolate chimeric protein with specific binding activity. A library may comprise a chimeric protein of any one of the first to the fourteenth aspects, each chimeric protein in the library comprising:

(i) a scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located in the scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the scaffold of the chimeric proteins in said library is diverse.

Chimeric proteins as described herein may be used to produce libraries. A suitable library may be screened in order to identify and isolate chimeric protein with specific binding activity. CKS Scaffold Libraries

In some embodiments of the first aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a CKS scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at one or more of the first loop, second loop, third loop or helical region of the CKS scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the CKS scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric proteins in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand in one of the first loop, second loop, third loop or helical region of the CKS scaffolds in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the first aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the CKS scaffold. For example, a library may comprise chimeric proteins of the first aspect, each chimeric protein in the library comprising:

(i) a CKS scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein said constant peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the diverse peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse. In some embodiments, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a CKS scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the CKS scaffold. For example, a library may comprise chimeric proteins of the first aspect, each chimeric protein in the library comprising:

(i) a CKS scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the first aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an CKS scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in one of the first loop, second loop, third loop or helical region of the CKS scaffold,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

Coiled-coil Scaffold Libraries

In some embodiments of the second aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID 1 or 3 or 5 or 6 or 7) of the coiled-coil scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the coiled- coil scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID 1 or 3 or 5 or 6 or 7) of the coiled-coil scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the second aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the second aspect, peptide ligands may be screened individually and a chimeric protein

assembled from the peptide ligands identified in different rounds of screening in the coiled- coil scaffold. For example, a library may comprise chimeric proteins of the second aspect, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule. In some embodiments of the second aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a coiled-coil scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the coiled-coil scaffold. For example, a library may comprise chimeric proteins of the second aspect, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands. An example of a schematic to generate such libraries is shown in Figure 1.

A library of the second aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a coiled-coil scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric protein.

Affibody Scaffold Libraries

In some embodiments of the third aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) an Affibody scaffold, and

(ii) one or more peptide ligands,

wherein said peptide ligands are located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22;

between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one of the peptide ligands in the Affibody scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric proteins in the library of the third aspect may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37;

between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53 of the Affibody scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the third aspect, peptide ligands may be screened individually and a chimeric proteins assembled from the peptide ligands identified in different rounds of screening in the Affibody scaffold. For example, a library may comprise chimeric proteins of the third aspect, each chimeric protein in the library comprising:

(i) an Affibody scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse. In some embodiments of the third aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the third aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in an Affibody scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the Affibody scaffold. For example, a library may comprise chimeric proteins of the third aspect, each chimeric protein in the library comprising:

(i) an Affibody scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 534) of the Affibody scaffold of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands. A schematic for generation of such a library is shown in Figure 1.

A library of the third aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Affibody scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein. Trefoil Scaffold Libraries

In some embodiments of the fourth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Trefoil scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the trefoil scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the a position between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of the Trefoil scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the fourth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the fourth aspect, peptide ligands may be screened individually and a chimeric protein

assembled from the peptide ligands identified in different rounds of screening in the Trefoil scaffold. For example, a library may comprise chimeric proteins of the fourth aspect, each chimeric protein in the library comprising:

(i) a Trefoil scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the fourth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the fourth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a Trefoil scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the Trefoil scaffold. For example, a library may comprise chimeric proteins of the fourth aspect, each chimeric protein in the library comprising:

(i) a Trefoil scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein, wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands. A schematic for generation of such a library is shown in Figure 1.

A library of the fourth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising (i) a Trefoil scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at the a position between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78 of the Trefoil scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

PDZ Scaffold Libraries

In some embodiments of the fifth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a PDZ scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the PDZ scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the positions between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the PDZ scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the fifth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library. In some embodiments of the fifth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the PDZ scaffold. For example, a library may comprise chimeric proteins of the fifth aspect, each chimeric protein in the library comprising:

(i) a PDZ scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the fifth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the fifth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a PDZ scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the PDZ scaffold. For example, a library may comprise chimeric proteins of the fifth aspect, each chimeric protein in the library comprising:

(i) a PDZ scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein, wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands. A schematic for generation of such a library is shown in Figure 1. A library of the fifth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an PDZ scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at one of the first, second, third, fourth loops and a first helix (for example between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

Ubiquitin or Ubiquitin-like Scaffold Libraries

In some embodiments of the sixth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at first, second, third loops and a first helix (for example at positions between residues 8 to 9;

between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305) of the Ubiquitin scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the Ubiquitin scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305 of the Ubiquitin scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the sixth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the sixth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the Ubiquitin scaffold. For example, a library may comprise chimeric proteins of the sixth aspect, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305) of the Ubiquitin scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the sixth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the sixth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in an Ubiquitin scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the Ubiquitin scaffold. For example, a library may comprise chimeric proteins of the sixth aspect, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands. An example of a schematic to generate such libraries is shown in Figure 1.

A library of the sixth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Ubiquitin scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

GB1 Scaffold Libraries

In some embodiments of the seventh aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a GB1 scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the GB1 scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric proteins in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand. In some embodiments of the seventh aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the seventh aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the GB1 scaffold. For example, a library may comprise chimeric proteins of the seventh aspect, each chimeric protein in the library comprising:

(i) a GB1 scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the seventh aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the seventh aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a GB1 scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the GB1 scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a GB1 scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the seventh aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a GB1 scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

WW Scaffold Libraries

In some embodiments of the eighth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a WW scaffold, and

(ii) one or more peptide ligands, wherein each said peptide ligand is located in the first loop or second loop of the WW scaffold or the helical region, if present; wherein at least one amino acid residue in at least one of the peptide ligands in the WW scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand in a loop of the WW scaffold in the library or a helical region if present, may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the eighth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library. In some embodiments of the eighth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the VWV scaffold. For example, a library may comprise chimeric proteins of the eighth aspect, each chimeric protein in the library comprising:

(i) a VWV scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the eighth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the eighth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a VWV scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the VWV scaffold. For example, a library may comprise chimeric proteins of the eighth aspect, each chimeric protein in the library comprising:

(i) a VWV scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein respectively,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the eighth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an VWV scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in the first loop or second loop of the VWV scaffold or the helical region, if present,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

Fibritin Scaffold Libraries

In some embodiments of the ninth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a Fibritin scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located in the coiled-coil subdomain and/or the disordered region of the Fibritin scaffold, wherein at least one amino acid residue in at least one of the peptide ligands in the Fibritin scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the coiled-coil subdomain or disordered region of the Fibritin scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the ninth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the ninth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the Fibritin scaffold. For example, a library may comprise chimeric proteins of the ninth aspect, each chimeric protein in the library comprising:

(i) a Fibritin scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein the constant peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the diverse peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the ninth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the ninth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a Fibritin scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the Fibritin scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a Fibritin scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the ninth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Fibritin scaffold; and

(ii) one or more peptide ligands, said peptide ligands being located in the coiled-coil subdomain of the Fibritin scaffold, the disordered region or both,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein. aPP Scaffold Libraries

In some embodiments of the tenth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) an aPP scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located in the loop or the helical region of the aPP scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the aPP scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand in the loop or helical region of the aPP scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the tenth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the tenth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the aPP scaffold. For example, a library may comprise chimeric proteins of the tenth aspect, each chimeric protein in the library comprising:

(i) an aPP scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein the constant peptide ligand is located at one of the loop and helical region of the aPP scaffold and the diverse peptide ligand is located at the other of the loop and helical region of the aPP scaffold.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse. In some embodiments of the tenth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the tenth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in an aPP scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the aPP scaffold. For example, a library may comprise chimeric proteins of the tenth aspect, each chimeric protein in the library comprising:

(i) an aPP scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule,

wherein the first peptide ligand is located at one of the loop and helical region of the aPP scaffold and the second peptide ligand is located at the other of the loop and helical region of the aPP scaffold,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the tenth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an aPP scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in the loop or helical region of the aPP scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric proteins.

Fibronectin Scaffold Libraries

In some embodiments of the eleventh aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a fibronectin scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located in one or more of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the fibronectin scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand in a loop of the fibronectin scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the eleventh aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the eleventh aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the fibronectin scaffold. For example, a library may comprise chimeric proteins of the eleventh aspect, each chimeric protein in the library comprising:

(i) a fibronectin scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein the constant peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the diverse peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the eleventh aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the eleventh aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a fibronectin scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the fibronectin scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a fibronectin scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the eleventh aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a fibronectin scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in one of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

Zn Finger Scaffold Libraries

In some embodiments of the twelfth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a Zn finger scaffold, and

(ii) one or more peptide ligands, wherein said peptide ligands are located in one or more of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein at least one amino acid residue in at least one of the peptide ligands in the Zn finger scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population. For example, 1 to n positions within a peptide ligand in the first loop, second loop or helical region of the Zn finger scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the twelfth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the twelfth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the Zn finger scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a Zn finger scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

wherein the constant peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the diverse peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the twelfth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the twelfth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a Zn finger scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the Zn finger scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a Zn finger scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the twelfth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Zn finger scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

SH3 Scaffold Libraries.

In some embodiments of the thirteenth aspect, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) an SH3 scaffold, and

(ii) one or two or more peptide ligands, said peptide ligands being located in one or two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein

wherein at least one amino acid residue in at least one of the peptide ligands in the SH3 scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the SH3 of the SH3 scaffold of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments of the thirteenth aspect, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments of the thirteenth aspect, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the SH3 scaffold. For example, a library may comprise chimeric proteins of the thirteenth aspect, each chimeric protein in the library comprising:

(i) an SH3 scaffold; and

(ii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,

said peptide ligands being located in one, two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein of the SH3 scaffold of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments of the thirteenth aspect, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments of the thirteenth aspect, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in an SH3 scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the SH3 scaffold. For example, a library may comprise chimeric proteins of the thirteenth aspect, each chimeric protein in the library comprising:

(i) an SH3 scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, said peptide ligands being located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library of the thirteenth aspect may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an SH3 scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in one of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population,

thereby producing a library of chimeric protein.

Cystine knot scaffold libraries

In some embodiments of the fourteenth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a cystine knot scaffold, and

(iii) one or more peptide ligands located at one or more of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein wherein at least one amino acid residue in at least one of the peptide ligands in the cystine knot scaffold of the chimeric proteins in said library is diverse.

The residues at one or more positions in a peptide ligand of the chimeric protein in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to n positions within a peptide ligand at the cystine knot of the chimeric protein in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments, one or two peptide ligands in the chimeric proteins in the library may be diverse. For example, the sequence of the one or two peptide ligands may be different in different chimeric proteins in library.

In some embodiments, peptide ligands may be screened individually and a chimeric protein assembled from the peptide ligands identified in different rounds of screening in the cystine knot scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a cystine knot scaffold; and

(iii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library, wherein

the constant peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the diverse peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold.

At least one amino acid residue in the diverse peptide ligands in said library may be diverse.

In some embodiments, a diverse peptide ligand in the chimeric proteins of the library may comprise different peptide sequences which bind to the same target molecule.

In some embodiments, different combinations of a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule may be screened individually in a cystine knot scaffold to identify a combination of peptide ligands that bind the first and second target molecules in the cystine knot scaffold. For example, a library may comprise chimeric protein, each chimeric protein in the library comprising:

(i) a cystine knot scaffold; and

(ii) a first peptide ligand for a first target molecule and a second peptide ligand for a second target molecule, wherein the first peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the second peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold,

wherein the first and second peptide ligands are diverse in the chimeric proteins in the library and the chimeric proteins in the library comprise different combinations of said first and second peptide ligands.

A library may be produced by a method comprising:

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a cystine knot scaffold; and

(iii) one or more peptide ligands located at one or more of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric protein.

Expression of Chimeric Protein Libraries

The population of nucleic acids may be provided by a method comprising inserting a first population of nucleic acids encoding a diverse peptide ligand into a second population of nucleic acids encoding scaffolds according to any one of the first to the fourteenth aspects, optionally wherein the first and second nucleic acids are linked with a third population of nucleic acids encoding linkers of up to 10 amino acids.

The nucleic acids may be contained in vectors, for example expression vectors. Suitable vectors include phage-based or phagemid-based phage display vectors.

The nucleic acids may be recombinantly expressed in a cell or in solution using a cell-free in vitro translation system such as a ribosome, to generate the library. In some preferred embodiments, the library is expressed in a system in which the function of the chimeric protein enables isolation of its encoding nucleic acid. For example, the chimeric protein may be displayed on a particle or molecular complex to enable selection and/or screening. In some embodiments, the library of chimeric protein may be displayed on beads, cell-free ribosomes, bacteriophage, prokaryotic cells or eukaryotic cells. Alternatively, the encoded chimeric protein may be presented within an emulsion where activity of the chimeric protein causes an identifiable change. Alternatively, the encoded chimeric protein may be expressed within or in proximity of a cell where activity of the chimeric protein causes a phenotypic change or changes in the expression of a reporter gene.

Preferably, the nucleic acids are expressed in a prokaryotic cell, such as E coli. For example, the nucleic acids may be expressed in a prokaryotic cell to generate a library of recombine binding proteins that is displayed on the surface of bacteriophage. Suitable prokaryotic phage display systems are well known in the art, and are described for example in Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001 , ISBN: 3540413545, W092/01047, US5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471 , US6172197, US6225447, US6291650, US6492160 and US6521404. Phage display systems allow the production of large libraries, for example libraries with 10 ⁸ or more, 10 ⁹ or more, or 10 ¹⁰ or more members.

In other embodiments, the cell may be a eukaryotic cell, such as a yeast, insect, plant or mammalian cell.

A diverse sequence as described herein is a sequence which varies between the members of a population i.e. the sequence is different in different members of the population. A diverse sequence may be random i.e. the identity of the amino acid or nucleotide at each position in the diverse sequence may be randomly selected from the complete set of naturally occurring amino acids or nucleotides or a sub-set thereof. Diversity may be introduced into the peptide ligand using approaches known to those skilled in the art, such as oligonucleotide-directed mutagenesis ²² , Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al. , 2001 , Cold Spring Harbor Laboratory Press, and references therein).

Diverse sequences may be contiguous or may be distributed within the peptide ligand.

Suitable methods for introducing diverse sequences into peptide ligand are well-described in the art and include oligonucleotide-directed mutagenesis (see Molecular Cloning: a

Laboratory Manual: 3rd edition, Russell et al., 2001 , Cold Spring Harbor Laboratory Press, and references therein). For example, diversification may be generated using oligonucleotide mixes created using partial or complete randomisation of nucleotides or created using codons mixtures, for example using trinucleotides. Alternatively, a population of diverse oligonucleotides may be synthesised using high throughput gene synthesis methods and combined to create a precisely defined and controlled population of peptide ligands.

Alternatively,“doping” techniques in which the original nucleotide predominates with alternative nucleotide(s) present at lower frequency may be used.

Preferably, the library is a display library. The chimeric protein in the library may be displayed on the surface of particles, or molecular complexes such as beads, for example, plastic or resin beads, ribosomes, cells or viruses, including replicable genetic packages, such as yeast, bacteria or bacteriophage (e.g. Fd, M13 or T7) particles, viruses, cells, including mammalian cells, or covalent, ribosomal or other in vitro display systems.

Techniques for the production of display libraries, such as phage display libraries are well known in the art. Each particle or molecular complex may comprise nucleic acid that encodes the chimeric protein that is displayed by the particle.

In some preferred embodiments, the chimeric proteins in the library are displayed on the surface of a viral particle such as a bacteriophage. Each chimeric protein in the library may further comprise a phage coat protein to facilitate display. Each viral particle may comprise nucleic acid encoding the chimeric protein displayed on the particle. Suitable viral particles include bacteriophage, for example filamentous bacteriophage such as M13 and Fd. Suitable methods for the generation and screening of phage display libraries are well known in the art. Phage display is described for example in W092/01047 and US patents

US5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471 , US6172197, US6225447, US6291650, US6492160 and

US6521404.

Screening Chimeric Protein Libraries

Libraries as described herein may be screened for chimeric proteins which display binding activity, for example binding to a target molecule. Binding may be measured directly or may be measured indirectly through agonistic or antagonistic effects resulting from binding. A method of screening may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a scaffold according to any one of the first to the fourteenth aspects; and

(ii) one or more peptide ligands, each said peptide ligand being located in the scaffold,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the first aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a CKS scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in one of the first loop, second loop, third loop or helical region of the CKS scaffold,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the second aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a coiled-coil scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of SEQ ID 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the third aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) an Affibody scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID NO: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the fourth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a Trefoil scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the fifth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a PDZ scaffold; and (ii) one or more peptide ligands, each said peptide ligand being located at one of the first, second, third, fourth loops and a first helix (for example between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the sixth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a Ubiquitin scaffold; and

(ii) one or more peptide ligands, each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of any one of SEQ ID NOs: 291 , 293, 295, 297, 299, 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and (c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the seventh aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a GB1 scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the eighth aspect, a method of screening may comprise; (a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a VWV scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in the first loop or second loop of the VWV scaffold or the helical region, if present,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the ninth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a Fibritin scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in the disordered region of the Fibritin scaffold, the coiled-coil subdomain or both, wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and (c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the tenth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) an aPP scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in the loop or helical region of the aPP scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the eleventh aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a fibronectin scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in one of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and (c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the twelfth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a Zn finger scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and (c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the thirteenth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) an SH3 scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at one of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein, wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and (c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments of the fourteenth aspect, a method of screening may comprise;

(a) providing a library of chimeric protein, each chimeric protein in the library comprising;

(i) a cystine knot scaffold; and

(iii) one or more peptide ligands, each said peptide ligand being located at one of the first, second, third, fourth, fifth or sixth loops of the cystine knot scaffold of the chimeric protein,

wherein one or more residues of at least one peptide ligand are diverse in said library,

(b) screening the library for chimeric protein which display a binding activity, and

(c) identifying one or more chimeric protein in the library which display the binding activity.

In some embodiments, the chimeric proteins in the library may comprise one peptide ligand with at least one diverse amino acid residue. The library may be screened for peptide ligands that bind to a target molecule. For example, a first library may be screened for a first peptide ligand that binds to a first target molecule and a second library may be screened for a second peptide ligand that binds to a second target molecule.

The first and second peptide ligands are in different locations in the chimeric protein. In the CKS scaffold, they are not both in the first loop, second loop, third loop or helical region. For example, both ligands may not be in the same position corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3; residues 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3; residues 58 to 64 of SEQ ID NO: 1 and residues 58 to 64 of SEQ ID NO: 3 or residues 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3 in the CKS scaffold. In the coiled coli scaffold, they are not both in the first helix, second helix, or first loop. For example, both ligands may not be in the same position corresponding to residues 55 to 58; residues 16 to 54; or residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14 in the coiled-coil scaffold. In the Affibody scaffold, they are not both in the first, second, or third helices or first or second loops. For example, both ligands may not be in the same position corresponding to residues 20 to 22 of SEQ ID NO: 16 or SEQ ID NO: 18; residues 38 to 39 of SEQ ID NO: 16 and SEQ ID NO: 18; residues 5 to 19 of SEQ ID NO: 16 and SEQ ID NO: 18; residues 23 to 37 of SEQ ID NO: 16 and SEQ ID NO: 18; or residues 40 to 45 of SEQ ID NO: 16 and SEQ ID NO: 18 in the Affibody scaffold. In the Trefoil scaffold, they are not both in the first, second, third, or fourth loops. For example, both ligands may not be in the same position corresponding to residues 10 to 14; residues 23 to 28; residues 33 to 36 or residues 57 to 61 of any one of SEQ ID NOs: 54, 56 or 58 to 78 in the Trefoil scaffold. In the PDZ scaffold, they are not both in the first, second, third, fourth and fifth loops and first helix. For example, both ligands may not be in the same position corresponding to residues 20 to 24; residues 73 to 82; residues 12 to 18; residues 95 to 101 ; residues 34 to 35 of SEQ ID NO: 79 of the PDZ scaffold. In the Ubiquitin scaffold, they are not both in the first, second, or third loops or the first helix. For example, both ligands are not in the same position corresponding to residues 8 to 9; residues 23 to 33; residues 53 to 54; or residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305. In the GB1 scaffold, they are not both in the first, second, third, or fourth loop or the first helix. For example, both ligands may not be in the same position corresponding to residues 9 to 10; residues 18 to 21 ; residues 36 to 40; residues 46 to 49; or residues 22 to 35 of any one of SEQ ID NOs: 307, 309 and 311 in the GB1 scaffold. In the VWV scaffold, they are not both in the first loop, the second loop or the helical region. For example, both ligands may not be in the same position corresponding to residues 30 to 43; residues 51 to 53; or residues 61 to 63 of SEQ ID NO: 359 in the GB1 scaffold. In the Fibritin scaffold, they are not both in the coiled-coil subdomain or the disordered region. For example, both ligands may not be in the same position corresponding to residues 1 to 38 or residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 in the Fibritin scaffold. In the aPP scaffold, they are not both in the loop or both in the helical region. For example, both ligands may not be in the same position corresponding to residues 9 to 13 or residues 14 to 32 of SEQ ID NO: 412 or SEQ ID NO: 414 or 415 in the aPP scaffold. In the Fibronectin scaffold, they are not both in the first, second, third or fourth loop. For example, both ligands may not be in the same position corresponding to residues 14 to 15; residues 25 to 26; residues 43 to 44; or residues 81 to 82 of SEQ ID NO: 412 or SEQ ID NO: 418 or 420 in the Fibronectin scaffold. In the Zn finger scaffold, they are not both in the first loop, the second loop or the helical region. For example, both ligands may not be in the same position corresponding to residues 6 to 9; residues 11 to 12; or residues 17 to 28 of SEQ ID NO: 423 in the Zn finger scaffold. In the SH3 scaffold, they are not both in the first, second, third or fourth loop. For example, both ligands may not be in the same position

corresponding to residues 9 to 24; residues 31 to 35; residues 33 to 46; or residues 55 to 56 of SEQ ID NO: 653 in the SH3 scaffold. In the cystine knot scaffold, they are not both in the first loop, second loop, third loop, fourth loop, fifth loop or sixth loop. For example, both ligands may not be in the same position corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842; residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 842; residues 11 to 14 of SEQ ID NO: 840 or residues 11 to 16 of SEQ ID NO: 842; residue 16 of SEQ ID NO: 840 or residue 18 of SEQ ID NO: 842; residues 18 to 21 of SEQ ID NO: 840 or residues 20 to 23 of SEQ ID NO: 842; or residues 23 to 30 of SEQ ID NO: 840 or residues 25 to 31 of SEQ ID NO: 842 in the cystine knot scaffold.

First and second peptide ligands that bind to the first and second target molecules, respectively, are identified from the first and second libraries. The identified first and second peptide ligands may then be incorporated into a chimeric protein that binds to the first and second target molecules.

A first library may comprise chimeric proteins with a first diverse peptide ligand having at least one diverse amino acid residue. In some embodiments, the first library may comprise a diverse population of peptide ligand sequences. A first peptide ligand that binds to a target molecule may be identified from the first library. Chimeric protein comprising the first peptide ligand may be used to generate a second library comprising a second diverse peptide ligand having at least one diverse amino acid residue.

For example, a chimeric protein of the first aspect from the first library may be modified by addition of a second diverse peptide ligand to a CKS scaffold at a different site selected from the first loop, second loop, third loop and helical region. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 25 to 39; residues 46 to 54; residues 58 to 64; or residues 40 to 45 of SEQ ID NO: 840 in the CKS scaffold.

A chimeric protein of the second aspect from the first library may be modified by addition of a second diverse peptide ligand to a coiled-coil scaffold at a different site selected from the first helix, second helix and first loop. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 55 to 58; residues 16 to 54 or residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14 in the coiled-coil scaffold.

A chimeric protein of the third aspect from the first library may be modified by addition of a second diverse peptide ligand to an Affibody scaffold at a different site selected from the first, second, or third helices or first or second loops. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18, and 20 to 53 of the Affibody scaffold.

A chimeric protein of the fourth aspect from the first library may be modified by addition of a second diverse peptide ligand to a Trefoil scaffold at a different site selected from the first, second, third, or fourth loops. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 10 to 14; residues 23 to 28; residues 33 to 36 or residues 57 to 61 of any one of SEQ ID NOs: 54, 56 or 58 to 78 in the Trefoil scaffold.

A chimeric protein of the fifth aspect from the first library may be modified by addition of a second diverse peptide ligand to a PDZ scaffold at a different site selected from the first, second, third, fourth and fifth loops and first helix. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 20 to 24; residues 73 to 82; residues 12 to 18; residues 95 to 101 or residues 34 to 35 of SEQ ID NO: 79 in the PDZ scaffold.

A chimeric protein of the sixth aspect from the first library may be modified by addition of a second diverse peptide ligand to a Ubiquitin scaffold at a different site selected from the first, second, third loops and first helix. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 8 to 9; between residues 23 to 33; residues 53 to 54; and residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305 in the Ubiquitin scaffold.

A chimeric protein of the seventh aspect from the first library may be modified by addition of a second diverse peptide ligand to a GB1 scaffold at a different site selected from the first, second, third, or fourth loop or the first helix. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 9 to 10; residues 18 to 21 ; residues 36 to 40; residues 46 to 49; or residues 22 to 35 of any one of SEQ ID NOs: 307, 309 and 311 in the GB1 scaffold.

A chimeric protein of the eighth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different location in the VWV scaffold selected from the first loop, second loop, and helical region. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 30 to 43; residues 51 to 53; or residues 61 to 63 of SEQ ID NO: 359 in the V V scaffold.

A chimeric protein of the ninth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different location in the Fibritin scaffold selected from the coiled-coil subdomain and disordered region. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 1 to 38 or residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 in the Fibritin scaffold.

A chimeric protein of the tenth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different location in the aPP scaffold selected from the loop and the helical region. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 9 to 13 or residues 14 to 32 of SEQ ID NO: 412 or SEQ ID NO: 414 or 415 in the aPP scaffold.

A chimeric protein of the eleventh aspect from the first library may be modified by addition of a second diverse peptide ligand at a different location in the FN3 scaffold selected from the first, second, third and fourth loops. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 14 to 15;

residues 25 to 26; residues 43 to 44; or residues 81 to 82 of SEQ ID NO: 418 or 420 in the Fibronectin scaffold.

A chimeric protein of the twelfth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different site in the Zn finger scaffold selected from the first loop, second loop and helical region. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 6 to 9; residues 11 to 12; or residues 17 to 28 of SEQ ID NO: 423 in the Zn finger scaffold.

A chimeric protein of the thirteenth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different loop of the SH3 scaffold selected from the first, second, third and fourth loops. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 9 to 24; residues 31 to 35; residues 33 to 46; or residues 55 to 56 of SEQ ID NO: 653 in the SH3 scaffold.

A chimeric protein of the fourteenth aspect from the first library may be modified by addition of a second diverse peptide ligand at a different site selected from the first, second, third, fourth, fifth, and sixth loops. For example, the second diverse peptide ligand may be added at a different site selected from a position corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842; residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 842; residues 11 to 14 of SEQ ID NO: 840 or residues 11 to 16 of SEQ ID NO: 842; residue 16 of SEQ ID NO: 840 or residue 18 of SEQ ID NO: 842; residues 18 to 21 of SEQ ID NO: 840 or residues 20 to 23 of SEQ ID NO: 842; and residues 23 to 30 of SEQ ID NO: 840 or residues 25 to 31 of SEQ ID NO: 842 in the cystine knot scaffold.

A second peptide ligand that binds to the same or a different target molecule may be identified from the second library.

The use of separate libraries for each peptide ligand allows large numbers of different variants of each peptide ligand to be screened independently and then combined. For example, a phage library of 10 ⁸ -10 ¹² first peptide ligand variants may be combined with a phage library of 10 ⁸ -10 ¹² second peptide ligand variants and a phage library of 10 ⁸ -10 ¹² third peptide ligand variants. In some embodiments of the first to the fourteenth aspects, a phage library of 10 ⁸ 10 ¹² first peptide ligand variants may be combined with a phage library of 10 ⁸ - 10 ¹² second peptide ligand variants to generate a chimeric protein with first and second peptide ligands.

Certain scaffolds described herein, for example scaffolds of the fifth, sixth, tenth and thirteenth aspects, may display binding activity. In some embodiments of these aspects, a phage library of 10 ⁸ 10 ¹² helical peptide ligand variants may be combined with a phage library of 10 ⁸ 10 ¹² scaffold variants to generate a chimeric protein with helical and scaffold ligands. For example, in some embodiments of the sixth aspect, a phage library of 10 ⁸ -10 ¹² loop peptide ligand variants may be combined with a phage library of 10 ⁸ -10 ¹² ubiquitin domain variants to generate a chimeric protein with loop peptide and scaffold ligands.

In other embodiments, the chimeric proteins in the library may comprise a first peptide ligand for a first target molecule, which has at least one diverse amino acid residue, and a second peptide ligand for a second target molecule, which has at least one diverse amino acid residue. The library may be screened for peptide ligands that bind to the first and second target molecules. For example, the library may be screened for chimeric proteins comprising a first peptide ligand that binds to a first target molecule and a second peptide ligand that binds to a second target molecule.

Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cellular phenotype. The one or more identified or selected chimeric protein may be recovered and subjected to further selection and/or screening.

Chimeric proteins as described herein may be used to produce libraries comprising different combinations of peptide ligands grafted into a scaffold of any one of the first to the fourteenth aspects. The combinations of ligands may comprise first peptide ligands that bind to a member of a protein degradation pathway, such as an E3 ubiquitin ligase, and second peptide ligands that bind to a target molecule. A library may be screened in order to identify and isolate chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule, (ii) causing degradation of the target molecule in a cell through the protein degradation pathway.

A library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a scaffold of any one of the first to the fourteenth aspects;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein said first and second peptide ligands are located in different loops or helical regions of the scaffold,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Suitable chimeric proteins, target molecules and members of protein degradation pathways and examples of peptide ligands thereto are described elsewhere herein.

A schematic depiction of a library comprising different peptide ligands for first and second target molecules is shown in Figure 1. The matrix shown in Figure 1 is for use in targeting b- catenin for degradation. These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway. Each of the target-binding peptides is derived from a different protein that interacts with b-catenin (see Table 1 for examples of sequences). Each of the degradation pathway-binding peptides (referred to as“degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways);‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in the Table 1. The schematic illustrates the

combinatioral“plug-and-play” nature of these matrices, in terms of the ability to slot in any target-recruiting peptide and degradation-pathway-recruiting peptide. The other factor that can be varied in the matrix arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery. Once the matrix is constructed, it can then be screened in cell-based assay in order to identify the best combination of two peptides and their positions within the scaffold that induces the greatest reduction in target protein levels. The same panel of diverse degradation pathway components can be used for screen for degradation of any target.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins may comprise:

(i) a scaffold of any one of the first to the fourteenth aspects;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, wherein said first and second peptide ligands are located in different loops or helical regions of the scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

Different chimeric proteins in the library may comprise a peptide ligand for a different E3 ubiquitin ligase. For example, the chimeric proteins in the library may comprise peptide ligands for a panel of E3 ubiquitin ligases, each chimeric protein in the library comprising a peptide ligand for one of the E3 ubiquitin ligases in the panel.

Numerous E3 ubiquitin ligases are known in the art. A suitable panel of E3 ubiquitin ligases may for example, comprise two, three, four, five or more of Mdm2, SCF ^Sk P ² , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1-Cdt2, DDB1-Cul4, DDB1-Cul5, SOCS box-Cul5-SPSB2, SOCS box-Cul5-SPSB4, CHIP, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C and b-TRP. Examples of peptide ligands for E3 ubiquitin ligases are shown in Table 2.

The target molecule may be a target molecule as described above, for example, b-catenin, KRAS, or C-Myc. The chimeric proteins in the library may comprise different peptide ligands for the target molecule i.e. different chimeric proteins in the library may comprise different peptide ligands for the same target molecule. Each chimeric protein in the library may comprise a different peptide ligand for the target molecule. Examples of peptide ligands target molecules are shown in Table 1. For example, the target molecule may be b-catenin, KRAS, or C-Myc and the chimeric proteins in the library may comprise different peptide ligands for b-catenin, KRAS, or C-Myc, respectively. Examples of different peptide ligands for b-catenin, KRAS, and C-Myc are shown in Table 1.

A library may comprise chimeric proteins of the first aspect, each chimeric protein in the library comprising:

(i) a CKS scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the first aspect may comprise:

(i) a CKS scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and (iii) a second peptide ligand for a target molecule, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the first aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a CKS scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the first aspect may comprise; (a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a CKS scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, wherein said first peptide ligand is located at one of the first loop, second loop, third loop or helical region of the CKS scaffold and the second peptide ligand is located at another of the first loop, second loop, third loop or helical region of the CKS scaffold of the CKS scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the second aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different

combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the second aspect may comprise:

(i) a coiled-coil scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the second aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and (iii) a second peptide ligand for a target molecule, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the second aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a coiled-coil scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 58 or between residues 16 to 54 or between residues 59 to 83 of any one of SEQ ID NOs 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the third aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) an Affibody scaffold; (ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18, and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the third aspect may comprise:

(i) an Affibody scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18, and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the third aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an Affibody scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18, and 20 to 53) of the Affibody scaffold of the chimeric protein, wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the third aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an Affibody scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18, and 20 to 53) of the Affibody scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the fourth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Trefoil scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein, wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the fourth aspect may comprise:

(i) a Trefoil scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78). of the Trefoil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the fourth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Trefoil scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the fourth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Trefoil scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54, 56 or 58 to 78) of the Trefoil scaffold of the chimeric protein, wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the fifth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a PDZ scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at positions of the PDZ scaffold of the chimeric protein corresponding to positions between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the fifth aspect may comprise:

(i) a PDZ scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at positions of the PDZ scaffold of the chimeric protein corresponding to positions between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the fifth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a PDZ scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at positions of the PDZ scaffold of the chimeric protein corresponding to positions between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79, wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the fifth aspect may comprise; (a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a PDZ scaffold; (ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at positions of the PDZ scaffold of the chimeric protein corresponding to positions between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the sixth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first, second, third loops and a first helix (for example at positions corresponding to positions between residues 8 to 9; residues 23 to 33; residues 53 to 54; or residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the sixth aspect may comprise:

(i) a Ubiquitin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first, second, third loops and a first helix (for example at positions corresponding to positions between residues 8 to 9; residues 23 to 33; residues 53 to 54; or residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the sixth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first, second, third loops and a first helix (for example at positions corresponding to positions between residues 8 to 9; residues 23 to 33; residues 53 to 54; or residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the sixth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at first, second, third loops and a first helix (for example at positions corresponding to positions between residues 8 to 9; residues 23 to 33; residues 53 to 54; or residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the seventh aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a GB1 scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the seventh aspect may comprise:

(i) a GB1 scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric domain,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the seventh aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a GB1 scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the seventh aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a GB1 scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the eighth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a VWV scaffold; (ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the eighth aspect may comprise:

(i) a VWV scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the eighth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a VWV scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein said first and second peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the eighth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a VWV scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein said first and second peptide ligands are located in (a) the first and second loops,

(b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the ninth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Fibritin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the ninth aspect may comprise:

(i) a Fibritin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the ninth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Fibritin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold, wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the ninth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Fibritin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, wherein the first peptide ligand is located at one of the coiled-coil subdomain and disordered region of the Fibritin scaffold and the second peptide ligand located at the other of the coiled-coil subdomain and disordered region of the Fibritin scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the tenth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) an aPP scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, wherein the first peptide ligand is located in one of the loop and helical region of the aPP scaffold and the second peptide ligand is located in the other of the loop and helical region of the aPP scaffold,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the tenth aspect may comprise:

(i) an aPP scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the loop and helical region of the aPP scaffold and the second peptide ligand is located in the other of the loop and helical region of the aPP scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands. A method of screening a library of chimeric proteins of the tenth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an aPP scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the loop and helical region of the aPP scaffold and the second peptide ligand is located in the other of the loop and helical region of the aPP scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the tenth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an aPP scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the loop and helical region of the aPP scaffold and the second peptide ligand is located in the other of the loop and helical region of the aPP scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity. In some embodiments of the eleventh aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a fibronectin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the eleventh aspect may comprise:

(i) a fibronectin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the eleventh aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a fibronectin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the eleventh aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a fibronectin scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first, second, third and fourth loops of the fibronectin scaffold and the second peptide ligand is located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the twelfth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a Zn finger scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the twelfth aspect may comprise:

(i) a Zn finger scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the twelfth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Zn finger scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway, (c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the twelfth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Zn finger scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold, preferably one of the first loop and helical region, and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold, preferably the other of the first loop and helical region, of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the thirteenth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) an SH3 scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

said peptide ligands being located at two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins of the thirteenth aspect may comprise:

(i) an SH3 scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, said peptide ligands being located at two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins of the thirteenth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an SH3 scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway located at one of the first, second, third and fourth loops of the SH3 scaffold and

(iii) a second peptide ligand for a target molecule located at another of the first, second, third and fourth loops of the SH3 scaffold

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins of the thirteenth aspect may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an SH3 scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase located at one of the first, second, third and fourth loops of the SH3 scaffold, and

(iii) a second peptide ligand for a target molecule located at another of the first, second, third and fourth loops of the SH3 scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments of the fourteenth aspect, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a cystine knot scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the second peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold, wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

Suitable chimeric proteins, target molecules and members of protein degradation pathways and examples of peptide ligands thereto are described elsewhere herein.

A schematic depiction of a library comprising different peptide ligand for first and second target molecules is shown in Figure 1.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins may comprise:

(i) a cystine knot scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the second peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold, wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands.

A method of screening a library of chimeric proteins may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a cystine knot scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the second peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a cystine knot scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located at one of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold and the second peptide ligand is located at another of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold, wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

A method may further comprise identifying one or more combinations of first and second peptide ligands in chimeric proteins in the library which display the activity.

Binding may be determined by any suitable technique. For example, the library may be contacted with the target molecule under binding conditions for a time period sufficient for the target molecule to interact with the library and form a binding reaction complex with a least one member thereof. Binding conditions are those conditions compatible with the known natural binding function of the target molecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest.

The library may be contacted with the target molecule in the form of a heterogeneous or homogeneous admixture. Thus, the members of the library can be in the solid phase with the target molecule present in the liquid phase. Alternatively, the target molecule can be in the solid phase with the members of the library present in the liquid phase. Still further, both the library members and the target molecule can be in the liquid phase.

Suitable methods for determining binding of a chimeric protein to a target molecule are well known in the art and include ELISA, bead- based binding assays (e.g. using streptavidin- coated beads in conjunction with biotinylated target molecules, surface plasmon resonance, flow cytometry, Western blotting, immunocytochemistry, immunoprecipitation, and affinity chromatography. Alternatively, biochemical or cell-based assays, such as fluorescence- based or luminescence-based reporter assays may be employed.

Multiple rounds of panning may be performed in order to identify chimeric protein which display the binding activity. For example, a population of chimeric proteins enriched for the binding activity may be recovered or isolated from the library and subjected to one or more further rounds of screening for the binding activity to produce one or further enriched populations. Chimeric proteins which display binding activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated. In some embodiments, binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme. For example, the library may be screened by expressing the library in reporter cells and identifying one or more reporter cells with altered gene expression or phenotype. Suitable functional screening techniques for screening recombinant populations of chimeric proteins are well-known in the art

Chimeric proteins which display the binding activity may be further engineered to improve an activity or property or introduce a new activity or property, for example a binding property such as affinity and/or specificity, an in vivo property such as solubility, plasma stability, or cell penetration, or an activity such as increased neutralization of the target molecule and/or modulation of a specific activity of the target molecule or an analytical property. Chimeric proteins may also be engineered to improve stability, solubility or expression level.

Further rounds of screening may be employed to identify chimeric proteins which display the improved property or activity. For example, a population of chimeric proteins enriched for binding to the target molecule may be recovered or isolated from the library and subjected to one or more further rounds of screening for the improved or new property or activity to produce one or further enriched populations. Optionally, this may be repeated one or more times. Chimeric proteins which display the improved property or activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.

A chimeric protein of any one of the first to the fourteenth aspects may be encapsulated in a liposome, for example for delivery into a cell. Preferred liposomes include fusogenic liposomes. Suitable fusogenic liposomes may comprise a cationic lipid, such as 1 , 2- dioleoyl-3-trimethylammoniumpropane (DOTAP), and a neutral lipid, such as

dioleoylphosphatidylethanolamine (DOPE) for example in a 1 :1 (w/w) ratio. Optionally, a liposome may further comprise an aromatic lipid, such as DiO (3, 3'- dioctadecyloxacarbocyanine perchlorate), DiR (1 , 1’-dioctadecyl-3, 3, 3', 3'- tetramethylindotricarbocyanine iodide), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- sindacene-3-propionyl)-1 ,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine

(triethylammonium salt) (BODIPY FL-DHPE), and 2-(4,4-difluoro-5-methyl-4-bora-3a,4a- diazas-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-ph osphocholine (BODIPY- C12HPC) for example in a 0.1 :1 :1 (w/w) ratio relative to the neutral and cationic lipid.

Suitable techniques for the encapsulation of proteins in liposomes and their delivery into cells are established in the art (see for example, Kube et al Langmuir (2017) 33 1051-1059; Kolasinac et al (2018) Int. J. Mol. Sci. 19 346).

A method described herein may comprise admixing a chimeric protein or encoding nucleic acid as described herein with a solution of lipids, for example in an organic solvent, such as chloroform, and evaporating the solvent to produce liposomes encapsulating the chimeric protein. Liposome encapsulations comprising a chimeric protein of any one of the first to the fourteenth aspects are provided as an aspect of the invention.

A chimeric protein or encoding nucleic acid as described herein may be admixed with a pharmaceutically acceptable excipient. A pharmaceutical composition comprising a chimeric protein or nucleic acid as described herein and a pharmaceutically acceptable excipient is provided as an aspect of the invention.

The term“pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be“acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

The pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the chimeric protein into association with a carrier which may constitute one or more accessory ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Pharmaceutical compositions may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

A chimeric protein, encoding nucleic acid or pharmaceutical composition comprising the chimeric protein or encoding nucleic acid may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

Pharmaceutical compositions suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Pharmaceutical compositions suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other micro particulate systems which are designed to target the compound to cells, tissue or organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 mg/ml, for example, from about 10 ng/ml to about 1 mg/ml. The

formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

It will be appreciated that appropriate dosages of the chimeric protein, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of diagnostic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the imaging agent, the amount of contrast required, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of imaging agent and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the imaging agent at a site, such as a tumour, a tissue of interest or the whole body, which allow for imaging without causing substantial harmful or deleterious side-effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.

Chimeric proteins of any one of the first to the fourteenth aspects may be used in methods of diagnosis or treatment in human or animal subjects, e.g. human. Chimeric proteins for a target molecule may be used to treat disorders associated with the target molecule.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term“comprising” replaced by the term“consisting of” and the aspects and embodiments described above with the term“comprising” replaced by the term ’’consisting essentially of.

References to the positions of residues in proteins described herein are inclusive. For example, a position between residues Xi and X ₂ includes the positions of residues Xi and X ₂ , as well as any intervening residues.

Unless context dictates otherwise, residue X or x as used herein may be independently any amino acid (i.e. an X or x residue in a sequence herein may be any amino acid and may be a different amino acid to another X or x residue in the same sequence).

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification, as well as the applications to which priority is statemented are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example,“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

1.1 Large-scale protein purification (His-tagged) from E. coli

The pRSET B (His-tag) constructs are transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies are grown in 2TY media containing ampicillin (50 micrograms/mL) at 37 °C, 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures are then induced with IPTG (0.5 mM) for 16-20 h at 20 °C or 4 h at 37 °C. Cells are pelleted by centrifugation at 3000 g (4 °C, 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCI, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris is pelleted by centrifugation at 15,000 g at 4 °C for 45 min. Ni-NTA beads 50% bed volume (GE Healthcare) (5 mL) are washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCI) before the supernatant of the cell lysate is bound to them for 1 hr at 4 °C in batch. The loaded beads are washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non specific interaction of lysate proteins with the beads. Samples are eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a Hi Load 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCI) and proteins separated in isocratic conditions. Purity is checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure are pooled. Purified protein is flash-frozen and stored at -80 °C until further use. Concentrations are determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity is confirmed using mass spectrometry (MALDI). 1.2 Large-scale protein purification (heat treatment) from E. coli

Many of the chimeric proteins of any one of the first to the fourteenth aspects are thermally very stable, with melting temperatures above 80°C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65 °C for 20 min. Very few of the E. coli proteins will remain folded at such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins are removed by centrifugation, leaving 80- 90% pure sample of the desired protein. Constructs that fold reversibly can be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.

This approach allows the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is potentially scalable to industrial production and bioreactors.

1.3 Small-scale purification of His-tagged proteins for higher-throughput testing

Plasmids are transformed into E. coli C41 cells and plated overnight. 15 mis of 2TY medium (Roche) containing 50 micrograms/ml ampicillin is placed in each one f multiple 50 ml tubes. Several colonies are picked from the plates and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells are grown at 37 °C until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells are pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAse I treatment is used. The lysate is spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.

The supernatant is added to 100 mI bed volume of pre-washed Ni-NTA agarose beads. The subseguent affinity purification is performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used). The first ish contained 10% BugBuster® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCI. The three successive ishes had 30 mM of imidazole in the chosen buffer. Beads are washed thoroughly to remove the detergent present in the BugBuster® solution. Protein is eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of

Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole is removed using a NAP-5 disposable gel-filtration column (GE

Healthcare). 1.4 Competition fluorescence polarisation assay to measure the binding of grafted scaffold proteins to a target protein

To measure the binding of a grafted scaffold to a target protein, Competition FP can be performed using 384-well black opague optiplate microplates and a CLARIOstar microplate reader. The grafted scaffold protein is titrated into a solution containing a mixture of FITC- labelled peptide ligand and target binding partner (target protein). The prepared plates are incubated for 30 minutes at room temperature before readings are taken. The grafted scaffold is then titrated into the preformed FITC-peptide— target protein complex. A decrease in polarisation with increasing concentrations of grafted scaffold indicates displacement of FITC- peptide upon binding of the grafted scaffold to its target.

1.5 Isothermal Titration Calorimetry (ITC) to measure the binding of grafted scaffold

proteins to a target protein

ITC can be performed using a VP-ITC instrument (Microcal). Grafted scaffolds are dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCI, 0.5 mM TCEP. Dialysed target protein (200 mM) is titrated into the sample cell containing the grafted scaffold at 20 mM.

Injections of target protein into the cell are initiated with a 5 mI_ injection, followed by 29 injections of 10 mI_. The reference power is set at 15 pCal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data are fitted using the instrument software a one-site binding model.

1. 6 Cell culture

HEK293T cells are cultured in Dulbecco’s Modified Eagle’s Medium (Sigma Aldrich)

supplemented with 10% fetal bovine serum and penicillin/streptomycin (LifeTech) at 37°C with 5% CC>2 air supply.

1. 7 Cell transfection

HEK293T are seeded in 6- well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol.

1. 8 Western blot assay of target-protein engagement and of target-protein levels

Plasmid encoding the target protein (1 mg) alone and with plasmid encoding one of various target-specific grafted scaffolds (1 mg) is transfected in HEK293T cells in 6- well plates using Lipofectamine2000. After 48 hours of transfection, the cells are lysed in 200 mI_ of Laemmli buffer. After sample is boiled at 95°C for 20 min proteins are resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting is performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in target protein levels upon co-transfection with bifunctional grafted scaffolds are evaluated by the densitometry of the bands corresponding to the target protein normalised to actin levels using ImageJ. Co-immunoprecipitation can also be used to show that the grafted scaffold binds to the target protein and/or to the desired component of the degradation machinery.

1.9 Liposomal formulation and cytotoxicity assay

To make liposomal formulations of proteins (LFP), lipids (DOTAP (cationic): DOPE (neutral): DiR (aromatic) = 1 :1 :0.1 w/w) are dissolved in chloroform, and solvent is evaporated under vacuum overnight. Resulting mixed lipid cake is hydrated with 10 mM HEPES pH 7.4, containing 27 mM protein, so that the total lipid concentration is 4 mg/ml. This mixture is vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins are stored at 4°C until further use. To make empty liposomes (EL, empty liposomes without proteins), lipid cake is hydrated with 10 mM HEPES pH 7.4 without proteins.

An ATP assay is used to investigate whether there is any cytotoxicity associated with EL and LFP. In a typical procedure, 2 x 10 ⁵ HEK 293T cells/well in 500 mΐ. of Dulbecco’s Modified Eagles Medium (DM EM) supplemented with 10% fetal bovine serum are grown for 24 hours in a 24-well cell culture plate. Cells are incubated with liposome (EULFP)-media (DMEM without FBS) mix, having different volumes (0-60 mί) of EL and LFP, for 15 minutes at 37°C. After washing twice with 1x PBS, 500 mί of CellTiter-Glo ^® Reagent (Promega) is added and luminescence is measured using a microplate reader as par the manufacture’s protocol. Untreated cells are used as control. Data are obtained from triplicate samples, and the standard deviations are calculated from two independent experiments.

1.10 HiBiT split-luciferase assay

An alternative method for measuring target protein levels is the Nano-Glo® HiBiT Lytic Detection System from Promega Corporation. It is based on the split NanoLuc assay, which consists of a large N-terminal fragment (LgBiT) and a small C-terminal region (SmBiT). Five of the SmBiT amino acids have been replaced to produce the HiBiT (VSGWRLFKKIS) fragment, which has greater affinity for the LgBiT fragment and maintains NanoLuc luciferase activity. Either the HiBiT-tagged target DNA can be transient transfected or the endogenous target can be monitored by knock-in of the HiBiT Tag sequence using

CRISPR/Cas9 technology. Subsequent introduction of the complementary polypeptide, LgBiT, results in spontaneous and high affinity interaction between the HiBiT Tag and LgBiT to reconstitute the luminescent NanoBit® enzyme. Detection of tagged protein levels is possible from live or lysed cells.

Protein is introduced into HEK293T cells by either DNA transient transfection or

encapsulation within fusogenic liposomes. HEK293T cells are seeded into either 24-well or 96-well plates After 24 hours, DNA encoding the HiBiT-tagged target protein (20 ng for 96- well plate; 100 ng for 24-well plate) is transiently transfected into cells. Chimeric protein DNA (100 ng) is either transiently transfected into cells at the same time as HiBiT-target DNA transfection or encapsulated into liposomes and introduced 24 hours into the cells after transfection. Cells are treated with chimeric protein-containing liposomes for 15 minutes before 2 hours of incubation.

Nano-Glo® HiBiT Lytic Buffer (LgBiT protein (1 :100), Nano-Glo® HiBiT Lytic Substrate (1 :50) 1x PBS (1 :1)) is added to the cells 24 hours after transient transfection or 2 hours after liposomal treatment. The plates are shaken on an orbital shaker (1 ,000 rpm, 10 min) to ensure homogenous cell lysis and equilibration of LgBiT and HiBiT in the cell lysate. The luminescence measurements are performed in white Nunclon™ Delta 96-well plates at 25 °C using a CLARIOstar plate reader using a 460-480 emission filter.

2. Results

The biophysical properties of a grafted scaffold may be assessed as follows: The molar ellipticity at 222 nm (a measure of helical structure content) is monitored as a function of increasing temperature. A decrease in the molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. This thermal unfolding experiment is used to determine the melting temperature of the scaffold and thereby to assess whether or not the grafting process has had a detrimental effect on the

thermostability of the scaffold.

An alternative method to determine the thermodynamic stability of the proteins is to measure chemical-induced denaturation (either guanidine hydrochloride (GdnHCI) or urea) monitored by intrinsic protein fluorescence (tryptophan and tyrosine residues). Solutions are dispensed into Corning® 96-well, half-area, black polystyrene plates (CLS3993) with a Microlab ML510B dispenser (Hamilton) and measurements are carried out on a CLARIOstar Plate Reader (BMG Labtech). The buffer is added first into the wells, followed by 15 pi aliquots of protein stock. A stock solution of chemical denaturant (either 7 M GdnHCI or 9 M urea) is then dispensed into the wells to create a chemical-denaturant concentration gradient. 2.1. Demonstration of proteins with a single binding function grafted onto an alpha-helix

2.1.1 Preparation of a helix-grafted scaffold that binds to a target protein

First, the helix of a given protein that interacts with its target binding partner is mapped onto the heptad distribution. For example, the helix of SOS1 that interacts with KRAS was mapped (Margarit et al. 2003 Cell 112 5 685-695) onto the heptad distribution. The heptad positions within the stapled SOS1 helical peptide produced by Leshchiner et al. (PNAS 2015 1 12 (6) 1761-1766) and set the stapled side of the peptide to form the hydrophobic interface with the rest of the scaffold protein. The grafted scaffold may then be docked against the target protein using Haddock software (de Vries & Bonvin 2011 ; de Vries et al. 2010). Haddock is a data- driven docking algorithm that uses known information about the interaction for its calculations. The active (primary interaction residues) and the passive (5 A proximity to active) residues are extracted and inputted into the calculations. Docking is not necessary to validate helical grafted scaffold, and inspection of the structure of the helix-target protein structure and of the scaffold structure may be sufficient: The geometry of alpha-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand.

2.2. Demonstration of a grafted scaffold protein with a single binding function grafted onto an

2.2.1 Preparation of a scaffold comprising a grafted loop

First, a peptide ligand that binds to a given target protein is grafted onto the scaffold in a loop. Binding of the loop-grafted scaffold may be tested using ITC. ITC is particularly useful to assess these interactions, as it can measure the stoichiometry (n) of the interaction, and thus inform as to which loops (if there is more than one loop) are more or less accessible to the target protein, and can inform as to whether a multi-loop scaffold affords multivalency. An advantage of a multivalent grafted scaffold is that one may achieve an avidity effect. This is particularly useful where a target molecule has multiple domains that can be bound by a peptide ligand. Binding of a multivalent grafted scaffold to such a target protein would produce an increased binding affinity and a decreased off rate according to the number of repeats in the grafted scaffold, thus achieving an avidity effect.

2.2.2 Effect of introducing multivalency into a single binding function scaffold

The function of a multi-valent grafted scaffold containing variable numbers of the peptide ligand binding motif that binds to a given target protein can be tested using the same assays as for the mono-valent grafted scaffold. The results are used to assess whether increased potency can be achieved by increasing the valency. 2.2.3 A loop-grafted scaffold using a peptide ligand (Nrf) that binds to E3 ubiguitin ligase

Cul3-Keap1

Keapl is the substrate recognition subunit of the Cul3-Keap1 ubiguitin ligase. A Keapl-binding seguence (the published“degron” peptide seguence derived from the Keapl substrate Nrf2) can be inserted into the scaffold loop. Immunoprecipitation is used to confirm binding of the grafted scaffold to Keapl . ITC analysis is used to assess the affinity of the interaction.

2.3. Hetero-bifunctional scaffolds that direct target proteins for ubiguitination and subseguent degradation

The Wnt/p-catenin signalling pathway is deregulated in many cancers and in neurodegenerative diseases, and therefore b-catenin is an important drug target. There are a large number of known binding seguences (both helical and non-helical) for b-catenin that appear suitable for grafting onto the scaffold. There are also substrate-derived binding peptides (“degrons”) known for many E3 ubiguitin ligase, such as degrons for E3 ligases Cul3-Keap1 , Mdm2 and SCF ^Sk P ² . A bispecific grafted scaffold is constructed using a selected b-catenin-binding peptide and binding peptides specific for Cu3-Keap1 , Mdm2 and SCF ^Skp2 .

To test whether these bispecific grafted scaffolds are capable of directing b-catenin for ubiguitination and degradation, the plasmid encoding the hetero-bifunctional scaffold is transfected into HEK293T cells using Lipofectamine2000 together with HA-tagged b-catenin plasmid (using cells transfected with HA-tagged b-catenin plasmid alone as a control). After 48 hours of transfection, the cells are lysed, the sample is boiled and proteins are resolved by SDS- PAGE and immunoblotting is performed using anti-HA and anti-actin antibodies. Changes in b- catenin levels are evaluated by the densitometry of the bands corresponding to HA-b-catenin normalised to actin levels. In this way, different combinations of b-catenin-binding peptides and degrons can be compared for their abilities to reduce the levels of b-catenin.

2.5 Using a delivery vehicle to introduce a grafted scaffold protein into cells

A grafted scaffold protein is encapsulated within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and then delivered into cells. Empty liposomes and liposomes encapsulating grafted scaffolds have been determined to be non-toxic to cells.

3. Measuring Endogenous 3-catenin Degradation

3.1 Generation of HiBiT tagged B-catenin MIA PaCa-2 cell line

B-catenin in MIA PaCa-2 cells was tagged with the HiBiT small peptide Tag by CRISPR editing and homology directed repair (HDR). Ribonuclearprotein complex of Cas9 enzyme and gRNA (mU ^* mG ^* mA ^* rCrCrU rGrUrA rArArU rCrArU rCrCrU rUrUrG rUrUrU rUrArG

stranded DNA HDR template

were aliquoted and frozen after 3 weeks. Aliquots were used on assays until passage 10.

3.2 Generation of HiBiT tagged KRAS HEK293 cell line

KRAS in HEK293 largeBIT cells was tagged with the HiBiT small peptide Tag by CRISPR editing and homology directed repair (HDR). Ribonuclearprotein complex of Cas9 enzyme and gRNA (Target sequence: CTTGTGGTAGTTGGAGCTGG) was prepared and electroporated into HEK293 largeBIT cells with a single stranded DNA HDR template

Single clones were selected after 4 weeks, Aliquots were used on assays until passage 10.

3.4 Reporter Assay for B-catenin Degradation

MIA PaCa-2 or HEK293 cells in which endogenous B-catenin has been tagged with the small peptide HiBiT (Mia PaCa-2 B-catenin HiBiT) were cultured in T175 flasks in DM EM 10% FCS at 37oC, 5% C02 and split 1 in 10 on reaching 90% confluency. Flasks were grown to approx. 80% confluency and cells were then split by gentle washing with 20 ml of PBS and incubation with 4ml of cell dissociation buffer for 5 minutes. Cells were counted and seeded at 5000 cells per well into replicate white solid bottom and black clear bottom plates in 100ul per well of DMEM 10%FCS. Cells were incubated overnight at 37°C, 5% C02.

Cells were transfected with 100ng per well of pcDNA 3.1 vector containing bispecific chimeric protein constructs using Lipofectamine 3000 using the manufacturer’s

recommended protocol (Thermofisher Scientific) or with 25nM siRNA (B-catenin targeted or Scramble control) using TransIT X2 using the manufacturer’s recommended protocol (Mirus Bio LLC).

24 hours after transfection the NanoGlo lytic detection assay was used to determine B- catenin-HiBiT levels. Plates were equilibrated to room temperature and 80mI of Nanoglo Lytic reagent was added to each well. After 10 minutes of shaking, luminescence was measured on a GloMax Discover plate reader with an integration time of 2 seconds.

3.5 Measurement of Chimeric Protein Expression in MIA PaCa-2 cells

MIA PaCa-2 cells were cultured and transfected with 100ng per well of pcDNA 3.1 vector containing chimeric protein constructs as described above.

24 hours after transfection, media was aspirated from the black clear bottom plates and cells were fixed with 4% PFA, quenched with 0.1M glycine in PBS, and permeabilized with IF blocking and permeabilization buffer (1* PBS, 5% FCS, 0.2% Saponin, 0.2uM filtered). Cells were stained with anti HA primary antibody (HA-Tag (C29F4) Rabbit mAb), followed Goat anti-Rabbit, Alexa Fluor® 555 secondary and Hoechst 33342 Nuclear counterstain. Wells were imaged immediately on an EVOS M5000 Fluorescence Microscope.

3.6 Reporter Assay for KRAS Degradation

HEK293 cells in which endogenous KRAS had been tagged with the small peptide HiBiTand the largeBiT fragment of NanoLuc was stably expressed ( HEK293 KRAS HiBiT large BiT) were cultured in T175 flasks in DM EM 10% FCS at 37oC, 10% C02 with 200pg/ml

Hygromycin selection and split 1 in 10 on reaching 90% confluency. Flasks were grown to approx. 80% confluency and cells were then split by gentle washing with 20 ml of PBS and incubation with 4ml of cell dissociation buffer for 5 minutes. Cells were counted and seeded at 5000 cells per well into white solid bottom plates in 100ul per well of DMEM 10%FCS. Cells were incubated overnight at 37°C, 10% CO2. Cells were transfected with 100ng per well of pcDNA 3.1 vector containing Trefoil constructs or with 25nM siRNA (KRAS targeted or Scramble control) using Lipofectamine 3000 using the manufacturers recommended protocol (Thermofisher Scientific). 24 hours after transfection the Nano-Glo Luciferase detection assay was used to determine KRAS-HiBiT levels. Plates were equilibrated to room temperature and 80ul of Nano-Glo® Luciferase Assay Reagent was added to each well.

After 5 minutes of shaking luminescence was measured on a GloMax Discover plate reader with an integration time of 2 seconds.

In other experiments, HEK293T cells were cultured in T75 flasks in DMEM 10% FBS, 5% pen/strep at 37°C, 5% CO2 and split 1 in 5 on reaching 90% confluency. Flasks were grown to approx. 80% confluency and cells were then split by gentle washing with 5 ml of PBS and incubation with 2ml of trypsin. Cells were counted and seeded at 15000 cells per well into 96-well cell culture white microplate (655083, Greiner) 100ul per well of DMEM 10%FBS, 5% pen/strep. Cells were incubated overnight at 37°C, 5% CO2. Cells were transfected with 20ng per well of pcDNA 3.1 vector containing KRAS4A-HiBiT and 100ng of empty vector (pcDNA3.1 empty) or CKS1 scaffold PPX constructs using Lipofectamine 2000 using the manufacturers recommended protocol. 24 hours after transfection the NanoGlo lytic detection assay (Promega) was used to determine KRAS4A-HiBiT levels. Plates were equilibrated to room temperature and 100ul of Nanoglo Lytic reagent was added to each well (after performing the CellTiter Fluor cell viability assay (G6080, Promega)). After 10 minutes of shaking luminescence was measured on a CLARIOstar plate reader.

3.7 Measurement of Chimeric Protein Expression in HEK293 cells

HEK293 cells were cultured and transfected with 100ng per well of pcDNA 3.1 vector containing chimeric protein constructs as described above. 24 hours after transfection, media was aspirated from the black clear bottom plates and cells were fixed with 4% PFA, quenched with 0.1 M glycine in PBS, and permeabilized with IF blocking and

permeabilization buffer (1* PBS, 5% FCS, 0.2% Saponin, 0.2uM filtered). Cells were stained with anti HA primary antibody (HA-Tag (C29F4) Rabbit mAb), followed Goat anti-Rabbit, Alexa Fluor® 555 secondary and Hoechst 33342 Nuclear counterstain. Wells were imaged immediately on an EVOS M5000 Fluorescence Microscope.

3.8 Western Blotting

HEK293 cells were cultured in T175 flasks in DMEM 10% FCS at 37oC, 10% C02 and split 1 in 10 on reaching 90% confluency. Flasks were grown to approx. 80% confluency and cells were then split by gentle washing with 20 ml of PBS and incubation with 4ml of cell dissociation buffer for 5 minutes. Cells were counted and seeded at 5000 cells per well into replicate white solid bottom and black clear bottom plates in 100ul per well of DMEM

10%FCS. Cells were incubated overnight at 37oC, 10% CO2. Cells were transfected with 100ng per well of pcDNA 3.1 vector containing chimeric protein constructs using

Lipofectamine 3000 using the manufacturers recommended protocol.

After 24 hours of transfection, the cells were lysed in 200 pL of RIPA buffer supplemented with protease inhibitors (ROCHE) and centrifuged at 12500rpm for 30 minutes at 4°C.

Supernatant was transferred in a clean tube, mixed with 100mM DTT, 1x laemmli buffer and denatured at 95°C for 5’. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. After blocking membrane with TBS, 0.2% Tween20, 5% dry non-fat milk, immunoblotting was performed using anti- b-catenin (ab16051 - Abeam) antibody. Changes in b-catenin levels were evaluated by the densitometric analysis after normalising total area to total protein and untreated cells. 3.9 Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) was used to monitor binding of PPX259 to Keap 1. Measurements were made at 25 °C using a MicroCal VP instrument (MicroCal,

Northampton, USA). Keapl protein was stored in a buffer containing 50 mM Tris-HCI, pH 7.5, 150 mM NaCI and 0.3 mM TCEP at 100 mM concentration.

PPX259 was dissolved in 100% DMSO to a final concentration of 2.5 mM. Prior to the ITC experiment, PPX259 was diluted to 70 mM using the buffer containing 50 mM Tris-HCI, pH 7.5, 150 mM NaCI and 0.3 mM TCEP. This resulted in a final DMSO concentration of 2.8%. Keapl protein was diluted into the same buffer, and DMSO was added to 2.8% to match the buffer composition of PPX259. Alternatively, PPX259 was diluted to 35 pM using the buffer containing 50 mM Tris-HCI, pH 7.5, 150 mM NaCI. This resulted in a final DMSO

concentration of 1.4%. Keapl protein was diluted into the same buffer, and DMSO was added to 1.4% to match the buffer composition of PPX259.

Both Keapl protein and PPX259 solutions were degassed prior to the titration experiment at 25 °C. The sample chamber and syringe were washed with water followed by buffer prior sample loading. 3 pL PPX259 (at 70 pM concentration) were injected followed by 46 injection steps of 6 pL into Keapl (10 pM). The concentration of Keapl was determined from an aliquot of the sample chamber to account for residual buffer after a wash that might have diluted the sample. The instrument was operated as per manufacturer's instructions, and data were analysed by MicroCal Origin software. Data were fitted to one-site binding model.

3.10 Competition Fluorescence Polarisation

PPX252 and PPX253 were dissolved in 100% (v/v) DMSO to 2 mM and 1 mM

concentrations, respectively. PPX253 did not fully dissolve and the undissolved pellet was centrifuged taking the supernatant. PPX252 and PPX253 were dispensed in a 1.5-fold dilution series across the wells of a black OptiPlate 384- F plate in assay buffer (phosphate buffered saline, 0.01% (v/v) Tween 20, 6% (v/v) DMSO). A complex of MDM2 and a

TAMRA-labelled p53 tracer peptide derived from p53 (Y. H. Lau et ai. , Chem. Sci. 2014, 5:

1804-1809) was prepared in assay buffer (without DMSO) and were added to the PPX samples to give a final concentration of 95 nM MDM2 and 50 nM TAMRA-labelled p53 tracer peptide in assay buffer (3% (v/v) DMSO). The plate was incubated for 30 minutes at 25 °C prior to measurement using a ClarioStar plate reader (BMG). Measurements were made with an excitation filter at 540 nm (bandwidth 20 nm) and emission filter at 590 nm

(bandwidth 20 nm) with a dichroic/low pass filter at 566 nm. The gain for the A channel was set at 1241 and gain for the B channel was set at 1281 , with the focal height set at 8.2 mm. Data were plotted in GraphPad Prism 7.0 and were fitted using competition binding equations (Z.-X Wang, FEBS Letters 1995 360: 111-114).

4. Results

4.1 CKS Scaffolds

4.1.1 B-catenin degradation by bispecific CKS1 constructs.

MIA PaCa-2 cells in which endogenous B-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or B-catenin targeted siRNA, or with CKS1 scaffold based constructs PPX69, PPX68, PPX70, PPX64, PPX65 and PPX66 (Figure 5A). The arrangement of these constructs is shown in Figure 5C and the sequences are provided in Table 4. The bispecific CKS1 based PPX65 was found to cause a 15.3% reduction in signal relative to lipofectamine only (LIPO), demonstrating its ability to degrade B-catenin. The bispecific CKS1 based PPX66 caused a 19.8% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade B-catenin. Positive control B-catenin targeted siRNA caused a 37.2% reduction in signal relative to scrambled RNA.

4.1.2 KRAS4A-HiBiT degradation by hetero-bifunctional CKS1 constructs.

HEK293T cells were co-transfected with KRAS4A tagged with the small peptide HiBiT and empty vector control (empty pcDNA3.1) or various CKS1 constructs (Figure 5B). PPX71 was found to show a 76% reduction in KRAS4A levels with respect to the vector control. PPX72 was found to show an 83% reduction in KRAS4A levels with respect to the vector control. PPX73 was found to show a 67% reduction in KRAS4A levels with respect to the vector control, PPX74 was found to show an 81% reduction in KRAS4A levels with respect to the vector control. PPX75 was found to show a 79% reduction in KRAS4A levels with respect to the vector control. PPX76 was found to show an 82% reduction in KRAS4A levels with respect to the vector control. PPX77 was found to show a 76% reduction in KRAS4A levels with respect to the vector control.

MIA PaCa-2 cells were transfected with Scrambled or B-catenin targeted siRNA, or with CKS1 scaffold based constructs PPX69, PPX68, PPX70, PPX64, PPX65 and PPX66 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 6). A box plot of mean intensity of HA staining per cell is shown in Figure 6 for each of the transfected constructs. HA was detected in cells transfected with PPX69, PPX68, PPX70, PPX64, PPX65 and PPX66 indicating that CKS1 based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.2 Coiled-coil scaffolds

4.2.1 B-catenin degradation by bispecific Coiled-coil constructs

MIA PACA 2 cells in which endogenous B-catenin was tagged with the small peptide HiBiT were transfected with Scrambled or B-catenin targeted siRNA, or with Coiled-coil scaffold based constructs PPX59, PPX58, PPX40, PPX41 , PPX42, PPX43, PPX44, PPX45, PPX46, PPX47, PPX48, PPX49, PPX50, PPX51 , PPX52, PPX53, PPX54, PPX55, PPX56 and PPX57 (Figure 10A). The arrangement of these constructs is shown in Figure 10C and the sequences are provided in Table 12. After 24 hours B-catenin abundance was quantified using the lytic HiBiT assay. The bispecific coiled-coil based PPX53 was found to cause a 46.5% reduction in signal relative to lipofectamine only (LIPO) (Figure 10A). The bispecific coiled-coil based PPX53 was found to cause a 42.6% reduction in signal relative to lipofectamine only (LIPO) and PPX41 was found to reduced signal by 30.1 % (Figure 10B). This demonstrates that bispecific degraders with coiled-coil scaffolds are capable of degrading B-catenin. Positive control B-catenin targeted siRNA caused a 65% reduction in signal in assay (10A) and a 61.3% reduction in assay (10B) relative to scrambled RNA.

4.2.2 Expression of Coiled-coil constructs in Mia PaCa 2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled or B-catenin targeted siRNA, or with Coiled-coil scaffold based constructs containing HA tags PPX59, PPX58, PPX40, PPX41 , PPX42, PPX43, PPX44, PPX45, PPX46, PPX47, PPX48, PPX49, PPX50, PPX51 , PPX52, PPX53, PPX54, PPX55, PPX56 and PPX57. After 24 hours cells were fixed, permeabilised and stained for HA Tag with Hoechst used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells from replicate experiments were analysed with Cell Profiler to quantify the intensity of HA staining in each cell. A box plot of mean intensity of HA staining per cell is shown in Figure 11 A for each of the transfected constructs. HA was detected in cells transfected with PPX59, PPX58, PPX48 and PPX41 by microscopy and in all Coiled-coil based polyproxins post analysis (Figure 11 B) indicating that Coiled-coil based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.2.3 Western Blotting of Coiled-coil Scaffold

The bifunctional Coiled-coil constructs PPX48 and PPX56 were found by western blotting to reduce b-catenin by 44% and 38% respectively after 24h in the HEK293 cells. This reduction is further increased at 48 hours, where it 87% and 59% was observed for PPX48 and PPX56 (Figures 11C and 11 D). These constructs did not show any reduction in Mia PaCa-2 (HiBiT assay Figures 10A and 10B) indicating activity may be cell type dependent.

4.3 Affibody Scaffolds

4.3.1 B-catenin degradation by bispecific Affibody constructs

MIA PaCa-2 cells in which endogenous B-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or B-catenin targeted siRNA, or with Affibody scaffold based constructs PPX86, PPX78, PPX80 and PPX81. The arrangement of these constructs is shown in Figure 16B and the sequences are provided in Table 16. After 24 hours B-catenin abundance was quantified using the lytic HiBiT assay (Figure 16A). The bispecific Affibody based scaffold PPX81 caused a reduction of 42.8% in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade B-catenin more effectively than its single function control (PPX78 17.7%). Positive control B-catenin targeted siRNA caused a 61.4% reduction in signal relative to scrambled RNA,

4.3.2 Expression of Affibody constructs in MIA PaCa-2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled or B-catenin targeted siRNA, or with Affibody scaffold based constructs PPX86, PPX78, PPX80 and PPX81 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were then analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 17). A box plot of mean intensity of HA staining per cell is shown in Figure 17 for each of the transfected constructs. HA was detected in cells transfected with PPX86, PPX78, PPX80 and PPX81 indicating that Affibody based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.4 Trefoil Scaffolds

4.4.1 B-catenin degradation by bispecific trefoil constructs

MIA PaCa-2 cells in which endogenous B-catenin has been tagged with the small peptide

HiBiT were transfected with Scrambled (SCRAM) or B-catenin targeted siRNA (siRNA), or with Trefoil scaffold based constructs PPX87, PPX93 and PPX88 (Figure 21 A). The arrangement of these constructs is shown in Figure 21 C and the sequences are provided in Table 20. After 24 hours B-catenin abundance was quantified using the lytic HiBiT assay.

The bispecific Trefoil based PPX88 was found to cause a 39% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade B-catenin more effectively than its single function controls (PPX87 21.3%). Positive control B-catenin targeted siRNA caused a 33% reduction in signal relative to scrambled RNA.

4.4.2 Expression of Trefoil constructs in Mia PaCa 2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled (scram) or b-catenin targeted siRNA (siRNA), or with Trefoil scaffold based constructs PPX87, PPX93 and PPX88 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell. A box plot of mean intensity of HA staining per cell is shown for each of the transfected constructs is shown in Figure 22. HA was detected in cells transfected with PPX87, PPX93 and PPX88 indicating that Trefoil based molecules are expressed in cells.

No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.4.3 KRAS degradation by bispecific trefoil constructs

HEK293 cells in which endogenous KRAS had been tagged with the small peptide HiBiT and the large BIT fragment of NanoLuc was stably expressed were transfected with Scrambled or KRAS targeted siRNA, or with Trefoil scaffold based constructs PPX91 , PPX89 and PPX93. After 24 hours KRAS abundance was quantified using the Nano-Glo Luciferase assay (Figure 21 B). The bispecific Trefoil based PPX91 was found to cause an 82% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade KRAS which was greater than either monospecific control (PPX89- 66% reduction, PPX93 - 55% reduction). Positive control KRAS targeted siRNA caused a 35% reduction in signal relative to scrambled RNA.

4.4.4 Expression of Trefoil constructs in HEK293 cells 48 hours after transfection

HEK293 cells were transfected Trefoil scaffold based constructs PPX89, PPX91 and PPX93 which contain HA tags. After 48 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoechst staining in transfected cells were produced and HA was detected in cells transfected with PPX89, PPX91 and PPX93 indicating that Trefoil based molecules are expressed in cells (not shown). Staining of untransfected cells was used for comparison to demonstrate that HA staining is specific for the transfected constructs.

4.5 PDZ Scaffolds

4.5.1 B-catenin degradation by bispecific PDZ constructs. MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with PDZ scaffold based constructs PPX94, PPX98, PPX95, PPX96 and PPX97. The arrangement of these constructs is shown in Figure 26C and the sequences are provided in Table 25. Bifunctional PDZ constructs tested do not show increased degradation of b-catenin relative to scaffold only control (PPX98; Figure 26A). Positive control b-catenin targeted siRNA caused a 61.4% reduction in signal relative to scrambled RNA.

4.5.2 Expression of PDZ constructs in MIA PaCa-2 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or KRAS targeted siRNA, or with PDZ scaffold based constructs PPX98, PPX94, PPX95 and PPX96 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti- HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell. A box plot of mean intensity of HA staining per cell is shown in Figure 27 for each of the

transfected constructs. HA was detected in cells transfected with PPX98, PPX94, PPX95 and PPX96 indicating that PDZ based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.5.3 KR \S degradation by bispecific PDZ constructs

HEK293 cells in which endogenous KRAS had been tagged with the small peptide HiBiT and the largeBIT fragment of NanoLuc was stably expressed were transfected with Scrambled or KRAS targeted siRNA, or with PDZ scaffold based constructs PPX99, PPX100, PPX101 and PPX102. After 24 hours KRAS abundance was quantified using the Nano-Glo Luciferase assay.

The bispecific PDZ based constructs PPX101 and PPX102 caused 77% and 61% reductions in signal respectively relative to lipofectamine only (LIPO) demonstrating their ability to degrade KRAS which was greater than their monospecific target binding controls (PPX99- 49% reduction, PPX100 - 45% reduction) (Figure 26B). Positive control KRAS targeted siRNA caused a 40% reduction in signal relative to scrambled RNA.

4.5.4 Expression of PDZ constructs in HEK293 cells 48 hours after transfection

HEK293 cells were transfected PDZ scaffold based constructs PPX99, PPX100, PPX101 and PPX102 which contain HA tags. After 48 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti- mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoechst staining in transfected cells were obtained (not shown). HA was detected in cells transfected with PPX99, PPX100, PPX101 and PPX102 indicating that PDZ based molecules are expressed in cells. Staining of untransfected cells was compared to demonstrate that HA staining is specific for the transfected constructs.

4.6 Ubiquitin or Ubiquitin-like Scaffolds

4.6.1 B-catenin deqradation by bispecific Ubiquitin and hPLIC constructs.

MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with Ub and hPLIC2 scaffold based constructs PPX103, PPX112, PPX104, PPX105, PPX5, PPX6 and PPX2

(Figure 31 A). The arrangement of these constructs is shown in Figure 31 B and the sequences are provided in Table 29. Positive control b-catenin targeted siRNA caused a 61.3% reduction in signal relative to scrambled RNA. The bispecific Ub based PPX104 caused a 46.5% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin more effectively than its single function control (PPX103 18.7%). The bispecific hPLIC2 based PPX2 caused a 38.7% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin more effectively than its single function control (PPX5 21.7%). Positive control b-catenin targeted siRNA caused a 52% reduction in signal relative to scrambled RNA.

4.6.2 Expression of Ub and hPLIC constructs in MIA PaCa-2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled or b-catenin targeted siRNA, or with hPLIC2 or RBD:Ub scaffold based constructs PPX103, PPX112, PPX104, PPX105, PPX5, PPX6 and PPX2 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 32). A box plot of mean intensity of HA staining per cell is shown for each of the transfected constructs. HA was detected in cells transfected with PPX103, PPX112, PPX104, PPX105, PPX5, PPX6 and PPX2 indicating that hPLIC2 and RBD:Ub based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.7 GB1 Scaffold

4.7.1 B-catenin deqradation by bispecific GB1 constructs.

HEK293 cells in which endogenous b-catenin as been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with GB1 scaffold based constructs PPX1 16, PPX117, PPX113, PPX1 14 and PPX1 15. The arrangement of these constructs is shown in Figure 36 B and the sequences are provided in Table 33. The bispecific GB1 based PPX113 caused a 39.3% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin more effectively than its single function control (PPX116) (Figure 36A). Positive control b-catenin targeted siRNA caused a 61.4% reduction in signal relative to scrambled RNA.

4.7.2 Expression of GB1 constructs in MIA PaCa-2 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or b-catenin targeted siRNA, or with GB1 scaffold based constructs PPX1 16, PPX117, PPX1 13, PPX114 and PPX115 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 37). A box plot of mean intensity of HA staining per cell is shown in Figure 37 for each of the transfected constructs. HA was detected in cells transfected with PPX116, PPX1 17, PPX113, PPX1 14 and PPX115 indicating that GBI based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.8 VWV Scaffolds

4.8.1 B-catenin degradation by bispecific VWV constructs

MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with VWV scaffold based constructs PPX123, PPX 124 and PPX122 (Figure 42A). The arrangement of these constructs is shown in Figure 42 B and the sequences are provided in Table 38. The bispecific VWV based PPX122 caused a 38.9% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin more effectively than its single function control (PPX123). Positive control b-catenin targeted siRNA caused a 61.3% reduction in signal relative to scrambled RNA.

4.8.2 Expression of VWV constructs in MIA PaCa-2 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or b-catenin targeted siRNA, or with VWV scaffold based constructs PPX123, PPX 124 and PPX122 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA tag. Hoechst was used as a nuclear counterstain. Images of HA and Hoechst staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 43). A box plot of mean intensity of HA staining per cell is shown in Figure 43 for each of the transfected constructs. HA was detected in cells transfected with PPX123, PPX 124 and PPX122 indicating that VWV based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.9 Fibritin Scaffolds

4.9.1 B-catenin degradation by bispecific Fibritin constructs

HEK293 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with Fibritin scaffold based constructs PPX132, PPX133, PPX129, PPX130 and PPX131 (Figure 47A).

The arrangement of these constructs is shown in Figure 47B and the sequences are provided in Table 41. The bispecific VWV based PPX122 caused a 25.9% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin. Positive control b-catenin targeted siRNA caused a 64.3% reduction in signal relative to scrambled RNA.

4.9.2 Expression of Fibritin constructs in MIA PaCa-2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled or KRAS targeted siRNA, or with Fibritin scaffold based constructs PPX132, PPX133, PPX129, PPX130 and PPX131 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 48). A box plot of mean intensity of HA staining per cell is shown in Figure 48 for each of the transfected constructs. HA was detected in cells transfected with PPX132, PPX133, PPX129, PPX130 and PPX131 indicating that Fibritin based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.10.1 B-catenin degradation by bispecific aPP and PPa constructs.

MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with aPP and PPa scaffold based constructs PPX161 , PPX162, PPX163, PPX154, PPX155, PPX156, PPX157, PPX158, PPX159, PPX160, PPX139 and PPX138 (Figure 50). The arrangement of these constructs is shown in Figure 51 and the sequences are provided in Table 45. The bispecific aPP based PPX138 caused a 58.1 % reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin. The bispecific PPa based

PPX159 caused a 34.9% reduction in signal relative to lipofectamine only (LIPO)

demonstrating its ability to degrade b-catenin. Positive control b-catenin targeted siRNA caused a 64% reduction in signal relative to scrambled RNA.

4.10.2 Expression of aPP and PPa constructs in MIA PaCa-2 24 hours after transfection.

MIA PaCa-2 cells were transfected with Scrambled or KRAS targeted siRNA, or with aPP and PPa scaffold based constructs PPX161 , PPX162, PPX163, PPX154, PPX155, PPX156, PPX157, PPX158, PPX159, PPX160, PPX139 and PPX138 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain to produce images of HA and Hoescht staining for transfected cells (not shown). HA was detected in cells transfected with PPX139 and PPX138 indicating that aPP and PPa based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.11 Fibronectin (FN3) Scaffolds

4.11.1 B-catenin degradation by bispecific FN3 constructs

HEK293 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with FN3 scaffold based constructs PPX143, PPX144 and PPX142 (Figure 53A). The arrangement of these constructs is shown in Figure 53 B and the sequences are provided in Table 48. The bispecific FN3 based PPX142 caused a 23.8% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin. Positive control b-catenin targeted siRNA caused a 64.3% reduction in signal relative to scrambled RNA.

4.11.2 Expression of FN3 constructs in 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or KRAS targeted siRNA, or with FN3 scaffold based constructs PPX143, PPX144 and PPX142 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 54). A box plot of mean intensity of HA staining per cell is shown in Figure 54 for each of the transfected constructs. HA was detected in cells transfected with PPX143 and PPX142 indicating that FN3 based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.12 Zn Finger Scaffolds

4.12.1 B-catenin degradation by bispecific ZF constructs

MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with ZF scaffold based constructs PPX148, PPX149, PPX145, PPX146 and PPX147 (Figure 57A). The arrangement of these constructs is shown in Figure 57 B and the sequences are provided in Table 51. The bispecific ZF based PPX147 caused a 31.3% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin. The bispecific ZF based PPX146 caused a 29.5% reduction in signal relative to lipofectamine only (LIPO) demonstrating its ability to degrade b-catenin. Positive control b-catenin targeted siRNA caused a 61.3% reduction in signal relative to scrambled RNA.

4.12.2 Expression of ZF constructs in MIAPACA 2 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or b-catenin targeted siRNA, or with ZF scaffold based constructs PPX148, PPX149, PPX145, PPX146 and PPX147 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary mouse anti-HA antibody and secondary donkey anti-mouse Alexa Fluor 647 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 58). A box plot of mean intensity of HA staining per cell is shown in Figure 58 for each of the transfected constructs. HA was detected in cells transfected with PPX146, PPX147, PPX148 and PPX149 indicating that ZF based molecules are expressed in cells.

No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein.

4.13 SH3 Scaffolds

4.13.1 B-catenin degradation by bispecific SH3 constructs

MIA PaCa-2 cells in which endogenous b-catenin has been tagged with the small peptide HiBiT were transfected with Scrambled or b-catenin targeted siRNA, or with SH3 scaffold based constructs. After 24 hours b-catenin abundance was quantified using the lytic HiBiT assay. Data from two independent experiments is shown in Figures 62A and 62 B. In a first assay, the bispecific SH3 based PPX21 and PPX33 showed a 34% and 50% respectively decrease over Lipososome alone control, demonstrating their ability to degrade b-catenin (Figure 62 A). Positive control b-catenin targeted siRNA caused a 61.3% reduction in signal relative to scrambled RNA. In a second assay, the bispecific SH3 based constructs PPX18 (55.5%), PPX19 (28.5%), PPX21 (65.3%), PPX22 (35.2%), PPX23 (62.5%), PPX25 (23.9%), PPX27 (34.4%), PPX28 (48.4%), PPX29 (39.9%) and PPX32 (28.8%) caused reduction in signal relative to lipofectamine only (LIPO) demonstrating the ability of SH3 based bifunctional constructs to degrade b-catenin (Figure 62 B). The arrangement of these constructs is shown in Figure 62 C and the sequences are provided in Table 55. Positive control b-catenin targeted siRNA caused a 40.6% reduction in signal relative to scrambled RNA.

4.13.2 B-catenin degradation induced by bifunctional hSH3 construct determined by

Western Blot in HEK293 cell line

Western blots were generated of b-catenin using HEK293 transfected with an hSH3 bifunctional construct PPX12. Total protein staining SDS-PAGE of HEK293 cell extract was observed after 24hrs (Figure 63A) and 48hrs (Figure 63B) transfection with PPX12 or untransfected controls. Total b-catenin western blot of HEK293 cell extract was observed fter 24hrs (Figure 63C) and 48hrs (Figure 63D) transfection with PPX12 or untransfected controls. These experiments showed that bifunctional hSH3 construct PPX12 reduced b- catenin by 55% at 24hrs and 80% at 48hrs in HEK293 cells.

4.13.3 Expression of SH3 constructs in MIA PaCa-2 24 hours after transfection

MIA PaCa-2 cells were transfected with Scrambled or b-catenin targeted siRNA, or with SH3 scaffold based constructs PPX34, 35, 14, 22, 33, 18, 21 , 23, 28 and 29 which contain HA tags. After 24 hours cells were fixed, permeabilised and stained for HA Tag using a primary rabbit anti-HA antibody and secondary goat anti-rabbit Alexa Fluor 555 conjugate. Hoescht was used as a nuclear counterstain. Images of HA and Hoescht staining for transfected cells were analysed with Cell Profiler to quantify the intensity of HA staining in each cell (Figure 64). A box plot of mean intensity of HA staining per cell is shown in Figure 64 for each of the transfected constructs. HA was detected in cells transfected with PPX34, 35, 14, 22, 33, 18, 21 , 23, 28 and 29 indicating that SH3 based molecules are expressed in cells. No HA was detected in siRNA treated cells as siRNA is not a HA tagged protein

4.14 Cystine Knot Scaffolds

4.14.1 Keapl binding of bispecific CK constructs

The binding of the cystine knot scaffold based construct PPX 259 to Keapl was measured using Isothermal titration calorimetry (ITC) in the presence of high concentration of reducing agent (0.3mM TCEP; Figure 65) and low concentration of reducing agent (0.015mM TCEP; Figure 66). A Kd of 18 nM for the binding of PPX 259 to Keapl was determined at both concentrations. 4.14.2 Competition fluorescence polarisation assay for binding of PPX252 and PPX253 to

MDM2

PPX252 and PPX253 were titrated into a solution of a complex between a TAMRA-labelled p53 tracer peptide and MDM2 N-terminal domain (residues 6-125). The apparent dissociation constants were found to be 13 ± 1 nM and 416 ± 84 nM for PPX252 and PPX253, respectively (Figure 67).

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TABLE 1

Target-binding ligands

TABLE 6

TABLE 7

TABLE 8

TABLE 18

(i) CKS Scaffolds

The following numbered statements relating to the first aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) a CKS scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or more of the first loop, second loop, third loop and helical region of the CKS scaffold of the chimeric protein.

2. A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3; the second loop is at a position corresponding to residues 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3; the third loop is at a position corresponding to residues 58 to 64 of SEQ ID NO: 1 and residues 58 to 64 of SEQ ID NO: 3 and the helical region is at a position corresponding to 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3.

6. A chimeric protein according to any one of the preceding statements wherein the CKS scaffold comprises the amino acid sequence of SEQ ID NO: 1 , 3 or 5 or a variant of any one of these.

7. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the first loop of the CKS scaffold.

8. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the second loop of the CKS scaffold

9. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the third loop of the CKS scaffold

10. A chimeric protein according to any one of statements 7 to 9 wherein the peptide ligand is connected to the loop by one or more additional residues.

11. A chimeric protein according to statement 10 wherein the peptide ligands are connected to the loops by a linker.

12. A chimeric protein according to any one of statements 7 to 12 the peptide ligand is connected to the loop is non-hydrophobic. 13. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the helical region of the CKS scaffold.

14. A chimeric protein according to statement 13 wherein the helical region is at a position corresponding to residues 40 to 45 of SEQ ID NO: 1 , 3 or 5.

15. A chimeric protein according to statement 13 or statement 14 wherein the peptide ligand in the helical region comprises an amino acid sequence of SEQ ID NO: 7 or a variant thereof.

16. A chimeric protein according to any one of statements 13 to 15 comprising a peptide ligand in the first loop of the CKS scaffold and a peptide ligand in the helical region of the CKS scaffold.

17. A chimeric protein according to any one of statements 13 to 15 comprising a peptide ligand in the second loop of the CKS scaffold and a peptide ligand in the helical region of the CKS scaffold.

18. A chimeric protein according to any one of statements 13 to 15 comprising a peptide ligand in the third loop of the CKS scaffold and a peptide ligand in the helical region of the CKS scaffold.

19. A chimeric protein according to any one of statements 1 to 18 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

20. A chimeric protein according to statement 19 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

21. A chimeric protein according to statement 20 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

22. A chimeric protein according to statement 21 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP. 23. A chimeric protein according to statement 20 wherein the cellular degradation pathway is cell mediated autophagy and optionally the peptide ligand is a heat shock cognate of 70kDa (Hsc70) peptide ligand.

24. A chimeric protein according to statement 20 wherein the cellular degradation pathway is lysosomal degradation and optionally the peptide ligand is ALIX, AP-1 , or A P-2.

25. A chimeric protein according to any one of statements 20 to 24 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

26. A chimeric protein according to any one of statements 20 to 25 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

27. A chimeric protein according to statement 26 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

28. A chimeric protein according to statement 26 or 27 comprising a peptide ligand having an amino acid sequence set out in Table 1

29. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

30. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule. 31. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

32. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

33. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

34. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

35. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

36. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule, and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 38. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule, and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

39. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

40. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 39.

41. An expression vector comprising a nucleic acid according to statement 40.

42. A host cell comprising an expression vector according to statement 41.

43. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 41 to produce the chimeric protein.

44. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 39; and

expressing said chimeric nucleic acid to produce the chimeric protein.

45. A method according to statement 44 further comprising inserting a second nucleic acid encoding a second peptide ligand into the second nucleic acid encoding a scaffold.

46. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein said peptide ligands are located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein

47. A method according to statement 46 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

48. A method according to statement 47 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

49. A library comprising chimeric proteins, each chimeric protein in the library comprising;

(i) a CKS scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

50. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in the two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different

combinations of said first and second peptide ligands.

51. A library according to statement 50 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

52. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a CKS scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second and third loops and the helical region of the CKS scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second and third loops and the helical region of the CKS scaffold.

53. A library according to any one of statements 49 to 52 wherein the chimeric proteins are according to any one of statements 1 to 39.

54. A library according to any one of statements 49 to 53 wherein the library is displayed on the surface of particles

55. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a CKS scaffold,

(ii) a peptide ligand located in one of the first, second and third loops and the helical region of the CKS scaffold, and;

(iii) a peptide ligand located in another of the first, second and third loops and the helical region of the CKS scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

56. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a CKS scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity. 57. A population of nucleic acids encoding a library according to any one of statements 49 to 54.

58. A method of producing a library comprising expressing a population of nucleic acids according to statement 57.

59. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a CKS scaffold,

(ii) a peptide ligand located in one of the first, second and third loops and the helical region of the CKS scaffold, and;

(iii) a peptide ligand located in another of the first, second and third loops and the helical region of the CKS scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

60. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 and a pharmaceutically acceptable excipient.

61. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 with a pharmaceutically acceptable excipient.

62. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41.

63. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector. 64. A chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 for use in a method of diagnosis or treatment in human or animal subject.

65. A chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 for use in the treatment of a disorder associated with the target molecule.

66. Use of a chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

67. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 comprising said nucleic acid to an individual in need thereof.

68. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 64 to 67 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(ii) Coiled-coil Scaffolds

The following numbered statements relating to the second aspect of the invention are part of the description.

1. A chimeric protein comprising;

(i) a Coiled-coil scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first helix, second helix, and first loop of the Coiled-coil scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 55 to 57 of SEQ ID NO: 8 or 10 or 12 to 14.

3 A chimeric protein according to any preceding statement wherein the first helix is at a position corresponding to residues 35 to 46 of SEQ ID NO: 8 or 10 or 12 to 14

4. A chimeric protein according to any preceding statement wherein the second helix is at a position corresponding to residues 66 to 77 of SEQ ID NO: 8 or 10 or 12 to 14.

5. A chimeric protein according to any preceding statement wherein the peptide ligand is immediately adjacent to a position corresponding to residues 16 or residue 83 of SEQ ID NO: 8 or 10 or 12 to 14.

6. A chimeric protein according to any preceding statement wherein the Coiled-coil scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 8 or 10 or 12 to 14, or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first helix and a second peptide ligand in the second helix of the Coiled-coil scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first helix and a second peptide ligand in the first loop of the Coiled-coil scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first helix and a second peptide ligand in the second helix of the Coiled- coil scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the second helix of the Coiled- coil scaffold. 14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the first helix of the Coiled-coil scaffold

15. A chimeric protein according to any one of statements 1 to 9, wherein chimeric protein further comprises a second loop, comprising a first peptide ligand in the second loop and a second peptide ligand in the first loop of the Coiled-coil scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR),

transcription factor, transcriptional regulator or bromodomain protein. 24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second helix that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the second helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25, wherein chimeric protein further comprises a third helix and a fourth helix, comprising a first peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third helix that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25, wherein chimeric protein further comprises a third helix and a fourth helix, comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the fourth helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25, wherein chimeric protein further comprises a third helix and a fourth helix, comprising a first peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third helix that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25, wherein chimeric protein further comprises a third helix and a fourth helix, comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the third helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25, wherein chimeric protein further comprises a third helix and a fourth helix, comprising a first peptide ligand in the second helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third helix that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain, oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Coiled-coil scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Coiled-coil scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth helices of the Coiled-coil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase. 47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a Coiled-coil scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth helices of the Coiled-coil scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third and fourth helices of the Coiled-coil scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Coiled-coil scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth helices of the Coiled-coil scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the Coiled-coil scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles 53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Coiled-coil scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth helices of the Coiled-coil scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third and fourth helices of the Coiled-coil scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Coiled-coil scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and fourth helices of the Coiled-coil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Coiled-coil scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth helices of the Coiled-coil scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth helices of the Coiled-coil scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(iii) Affibody Scaffolds

The following numbered statements relating to the third aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) an Affibody scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first helix is at a position corresponding to residues 5 to 19 of any one of SEQ ID NOs: 16, 18 or 20 to 53.

3 A chimeric protein according to any preceding statement wherein the second helix is at a position corresponding to residues 23 to 37 of any one of SEQ ID NOs: 16, 18 or 20 to 53.

4. A chimeric protein according to any preceding statement wherein the third helix is at a position corresponding to residues 40 to 56 of any one of SEQ ID NOs: 16, 18 or 20 to 53. 5. A chimeric protein according to any preceding statement wherein the first loop is at a position corresponding to residues 20 to 22 of any one of SEQ ID NOs: 16, 18 or 20 to 53.

6. A chimeric protein according to any preceding statement wherein the Affibody scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 16, 18 or 20 to 53 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first helix and a second peptide ligand in the second loop of the Affibody scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the Affibody scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first helix and a second peptide ligand in the second helix of the Affibody scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the third helix of the Affibody scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the second helix of the Affibody scaffold 15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third helix and a second peptide ligand in the first loop of the Affibody scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR),

transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2 or Table 7.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second helix that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third helix that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third helix that binds a target molecule. 32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the third helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second helix that binds a target molecule and a second peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second helix that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third helix that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain, oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38. 40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an Affibody scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an Affibody scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) an Affibody scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an Affibody scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the Affibody scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising; (i) an Affibody scaffold,

(ii) a first peptide ligand located in one of the first, second, third helices and first, second loops of the Affibody scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third helices and first, second loops of the Affibody scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an Affibody scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two or more of the first, second, third helices and first, second loops of the Affibody scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an Affibody scaffold,

(ii) a first peptide ligand located in one of the first, second, third helices and first, second loops of the Affibody scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third helices and first, second loops of the Affibody scaffold,

wherein the peptide ligands in said population are diverse, and (b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule. 64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(iv) Trefoil Scaffolds

The following numbered statements relating to the fourth aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) a Trefoil scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the Trefoil scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 10 to 14 of any one of SEQ ID NOs: 54, 56 or 58 to 78.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 23 to 28 of any one of SEQ ID NOs: 54, 56 or 58 to 78.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 33 to 36 of any one of SEQ ID NOs: 54, 56 or 58 to 78.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residues 57 to 61 of any one of SEQ ID NOs: 54, 56 or 58 to 78

6. A chimeric protein according to any preceding statement wherein the Trefoil scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 54, 56 or 58 to 78 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the Trefoil scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the Trefoil scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the fourth loop of the Trefoil scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the third loop of the Trefoil scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the fourth loop of the Trefoil scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the fourth loop of the Trefoil scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR),

transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2 or Table 7.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Trefoil scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Trefoil scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the Trefoil scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a Trefoil scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the Trefoil scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third and fourth loops of the Trefoil scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Trefoil scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the Trefoil scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the Trefoil scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Trefoil scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the Trefoil scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third and fourth loops of the Trefoil scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and (c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Trefoil scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and fourth loops of the Trefoil scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Trefoil scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the Trefoil scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the Trefoil scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

70. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

71. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

72. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

73. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

74. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

75. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 71 to 74 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(v) PDZ Scaffolds

The following numbered statements relating to the fifth aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) a PDZ scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third, fourth and fifth loops and a first helix of the PDZ scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein first loop is at a position corresponding to residues 20 to 24 of SEQ ID NO: 79; residues 18 to 54 of SEQ ID NO: 80; residues 19 to 22 of SEQ ID NO: 81 ; residues 13-17 of SEQ ID NO: 82; residues 13 to 23 of SEQ ID NO: 83; residues 20 to 24 of SEQ ID NO: 84; residues 10 to 15 of SEQ ID NO: 85; or residues 20 to 24 of SEQ ID NO: 87.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 51 to 56 of SEQ ID NO: 79; residues 59 to 65 of SEQ ID NO: 80; residues 30 to 42 of SEQ ID NO: 81 ; residues 23-34 of SEQ ID NO: 82; residues 32 to 34 of SEQ ID NO: 83; residues 30 to 38 of SEQ ID NO: 84; residues 21 to 31 of SEQ ID NO: 85; or residues 51 to 56 of SEQ ID NO: 87.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 69 to 72 of SEQ ID NO: 79; residues 76 to 81 of SEQ ID NO: 80; residues 51 to 54 of SEQ ID NO: 81 ; residues 42 to 44 of SEQ ID NO: 82; residues 52 to 56 of SEQ ID NO: 83; residues 45 to 49 of SEQ ID NO: 84; residues 46 to 52 of SEQ ID NO: 85; or residues 69 to 72 of SEQ ID NO: 87.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residues 57 to 62 of SEQ ID NO: 81 ; residues 50 to 55 of SEQ ID NO: 82; residues 64 to 71 of SEQ ID NO: 83; residues 52 to 62 of SEQ ID NO: 84; or residues 63 to 67 of SEQ ID NO: 85.

6. A chimeric protein according to any preceding statement wherein the PDZ scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 79 to 85 and 87 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the PDZ scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the PDZ scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the fourth loop of the PDZ scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the first helix of the PDZ scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the fourth loop of the PDZ scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the first helix of the PDZ scaffold 16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR),

transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39. 41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a PDZ scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and

expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a PDZ scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third .fourth loops and a first helix of the PDZ scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a PDZ scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement 47 wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different

combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a PDZ scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third , fourth loops and a first helix of the PDZ scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the PDZ scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a PDZ scaffold,

(ii) a first peptide ligand located in one of the first, second, third, fourth loops and a first helix of the PDZ scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third, fourth loops and a first helix of the PDZ scaffold, wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a PDZ scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a PDZ scaffold,

(ii) a first peptide ligand located in one of the first, second, third, fourth loops and a first helix of the PDZ scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third, fourth loops and a first helix of the PDZ scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(Vi) ubiquitin or ubiquitin-like domain scaffolds

The following numbered statements relating to the sixth aspect of the invention form part of the description

1. A chimeric protein comprising;

(i) a Ubiquitin scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, and third loops of the Ubiquitin scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 8 to 9 of any one of SEQ ID NOs: 291 , 293, 295, 297,299, 303 and 305.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 53 to 54 of any one of SEQ ID NOs: 291 , 293, 295,

297,299, 303 and 305.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 62 to 63 of any one of SEQ ID NOs: 291 , 293, 295,

297,299, 303 and 305.

5. A chimeric protein according to any preceding statement wherein the chimeric protein further comprises a first helix at a position corresponding to residues 23 to 33 of any one of SEQ ID NOs: 291 , 293, 295, 297,299, 303 and 305.

6. A chimeric protein according to any preceding statement wherein the Ubiquitin scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 291 , 293, 295,

297,299, 303 and 305 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the Ubiquitin scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the Ubiquitin scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the first helix of the Ubiquitin scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the first helix of the

Ubiquitin scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the first helix of the Ubiquitin scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the Ubiquitin scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2 or Table 7.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the first helix that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain, oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising; inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Ubiquitin scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Ubiquitin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third loops and the first helix of the Ubiquitin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a Ubiquitin scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third loops and the first helix of the Ubiquitin scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement 47 wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third loops and the first helix of the Ubiquitin scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Ubiquitin scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third loops and the first helix of the Ubiquitin scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third loops and the first helix of the Ubiquitin scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Ubiquitin scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the Ubiquitin scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third loops and the first helix of the Ubiquitin scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity. 54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Ubiquitin scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and the first helix of the Ubiquitin scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Ubiquitin scaffold,

(ii) a first peptide ligand located in one of the first, second, third loops and the first helix of the Ubiquitin scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the Ubiquitin scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof. 66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(vii) GB1 scaffold

The following numbered statements relating to the seventh aspect of the invention form part of the description

1. A chimeric protein comprising;

(i) a GB1 scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third , fourth loops and the first helix of the GB1 scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 9 to 10 of any one of SEQ ID NOs: 307; 309, 311 , and 313 to 348.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 18 to 21 of any one of SEQ ID NOs: 307; 309, 311 , and 313 to 348.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 36 to 40 of any one of SEQ ID NOs: 307; 309, 311 , and 313 to 348.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residues 46 to 49 of any one of SEQ ID NOs: 307; 309, 311 , and 313 to 348.

6. A chimeric protein according to any preceding statement wherein the GB1 scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 307; 309, 311 , and 313 to 348 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues. 8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the GB1 scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the GB1 scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the fourth loop of the GB1 scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the first helix of the GB1 scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first helix and a second peptide ligand in the fourth loop of the GB1 scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the first helix of the GB1 scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 3.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 2 or Table 7.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first helix that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a GB1 scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein. 44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a GB1 scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third , fourth loops and a first helix of the GB1 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a GB1 scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the GB1 scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third .fourth loops and a first helix of the GB1 scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a GB1 scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third, fourth loops and a first helix of the GB1 scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the GB1 scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a GB1 scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the GB1 scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third, fourth loops and a first helix of the GB1 scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a GB1 scaffold; (ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third, fourth loops and a first helix of the GB1 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a GB1 scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the GB1 scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third , fourth loops and a first helix of the GB1 scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

70. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

71. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

72. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

73. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

74. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

75. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 71 to 74 wherein the chimeric protein, nucleic acid or vector is encapsulated in a liposome. (viii) VWV scaffold

The following numbered statements relating to the eighth aspect of the invention form part of the description

1. A chimeric protein comprising;

(i) a VWV scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or both of the first and second loops of the VWV scaffold of the chimeric protein.

2. A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355, and residues 51 to 53 of SEQ ID NO: 359.

3. A chimeric protein according to statement 1 or statement 2 wherein the second loop is at a position corresponding to residues 23 to 25 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 29 to 31 of SEQ ID NO: 351 , residues 32 to 34 of SEQ ID NO: 353, residues 24 to 26 of SEQ ID NO: 355 and residues 61 to 63 of SEQ ID NO: 359.

4. A chimeric protein according to any one of the preceding statements wherein the VWV scaffold comprises the amino acid sequence of any one of SEQ ID NOs: 349, 351 , 353, 355, 357, or 359, an amino acid sequence set out in Tables 34 to 37, or a variant of any one of these.

5. A chimeric protein according to any one of the preceding statements wherein the peptide ligands are connected to the loops by one or more additional residues.

6. A chimeric protein according to any one of the preceding statements wherein the peptide ligands are connected to the loops by a linker.

7. A chimeric protein according to any one of the preceding statements wherein the one or more peptide ligands in the loops are non-hydrophobic.

8. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the first loop and a peptide ligand in the second loop of the VWV scaffold.

9. A chimeric protein according to any one of the preceding statements wherein the VWV scaffold further comprises a helical region (a helixWW scaffold), said helical region comprising a peptide ligand.

10. A chimeric protein according to statement 9 wherein the helical region is at a position corresponding to residues 30 to 43 of SEQ ID NO: 11.

11. A chimeric protein according to statement 9 or statement 10 wherein the VWV scaffold comprises an amino acid sequence of SEQ ID NO: 11 , an amino acid sequence set out in Table 6 or a variant thereof.

12. A chimeric protein according to any one of statements 9 to 11 wherein the peptide ligand in the helical of the VWV scaffold comprises an amino acid sequence of SEQ ID NO:

13 or a fragment or variant thereof.

13. A chimeric protein according to any one of statements 9 to 12 comprising a peptide ligand in the first loop of the VWV scaffold and a peptide in the helical region of the VWV scaffold.

14. A chimeric protein according to any one of statements 9 to 12 comprising a peptide ligand in the second loop of the VWV scaffold and a peptide ligand in the helical region of the VWV scaffold.

15. A chimeric protein according to any one of statements 1 to 14 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

16. A chimeric protein according to statement 15 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

17. A chimeric protein according to statement 16 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

18. A chimeric protein according to statement 17 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

19. A chimeric protein according to statement 16 wherein the cellular degradation pathway is cell mediated autophagy and optionally the peptide ligand is a heat shock cognate of 70kDa (Hsc70) peptide ligand.

20. A chimeric protein according to statement 16 wherein the cellular degradation pathway is lysosomal degradation and optionally the peptide ligand is ALIX, AP-1 , or A P-2.

21. A chimeric protein according to statement wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

22. A chimeric protein according to any one of statements 15 to 21 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

23. A chimeric protein according to statement 22 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

24. A chimeric protein according to statement 22 or 23 comprising a peptide ligand having an amino acid sequence set out in Table 1.

25. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

26. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

27. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

29. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the helical region that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the helical region that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

32. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 31.

33. An expression vector comprising a nucleic acid according to statement 32.

34. A host cell comprising an expression vector according to statement 33.

35. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 32 to produce the chimeric protein.

36. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a VWV scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 31 ; and expressing said chimeric nucleic acid to produce the chimeric protein.

37. A method according to statement 36 further comprising inserting a second nucleic acid encoding a second peptide ligand into the second nucleic acid encoding a WW scaffold.

38. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a WW scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein said peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the WW scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

39. A method according to statement 38 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

40. A method according to statement 39 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

41. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a WW scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the WW scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

42. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in the first and second loops, the first loop and the helical region or the second loop and the helical region of the WW scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

43. A library according to statement 42 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

44. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a VWV scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold.

45. A library according to any one of statements 41 to 44 wherein the chimeric proteins are according to any one of statements 1 to 31.

46. A library according to any one of statements 41 to 45 wherein the library is displayed on the surface of particles

47. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a VWV scaffold,

(ii) a peptide ligand located in one of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and (c) identifying one or more chimeric proteins in the library which display the binding activity.

48. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a VWV scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in the first and second loops, the first loop and the helical region or the second loop and the helical region of the VWV scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

49. A population of nucleic acids encoding a library according to any one of statements 41 to 46.

50. A method of producing a library comprising expressing a population of nucleic acids according to statement 49.

51. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a VWV scaffold,

(ii) a peptide ligand located in one of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold,

wherein the peptide ligands in said population are diverse, and (b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

52. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 and a pharmaceutically acceptable excipient.

53. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 with a pharmaceutically acceptable excipient.

54. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33.

55. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

56. A chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 for use in a method of diagnosis or treatment in human or animal subject.

57. A chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 for use in the treatment of a disorder associated with the target molecule.

58. Use of a chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

59. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 comprising said nucleic acid to an individual in need thereof.

60. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 56 to 58 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(ix) Fibritin scaffold

The following numbered statements relating to the ninth aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) a Fibritin scaffold,

(ii) first peptide ligand in the disordered region of the Fibritin scaffold; and

(iii) a second peptide ligand in the coiled-coil subdomain of the Fibritin scaffold.

2. A chimeric protein according to statement 1 wherein the coiled-coil subdomain is at a position corresponding to residues 1 to 38 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 15 of SEQ ID NO 365:

3. A chimeric protein according to any one of the preceding statements wherein the disordered region is at a position corresponding to residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 16 to 27 of SEQ ID NO: 365.

4. A chimeric protein according to any one of the preceding statements wherein the Fibritin scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 363, 365, 367, and 371-409 or a variant thereof.

5. A chimeric protein according to any one of the preceding statements wherein the first peptide ligand is connected to the disordered region by one or more additional residues.

6. A chimeric protein according to statement 5 wherein the first peptide ligand is connected to the disordered region by a linker.

7. A chimeric protein according to any one of the preceding statements wherein the first peptide ligand in the disordered region is non-hydrophobic. 8. A chimeric protein according to any one of the preceding statements wherein the second peptide ligand in the coiled-coil subdomain comprises SEQ ID NO: 7 or SEQ ID NO: 8 or a fragment thereof.

9. A chimeric protein according to any one of the preceding statements wherein the first peptide ligand binds one of a first and second target molecule and the second peptide ligand binds the other of the first and second target molecule.

10. A chimeric protein according to statement 9 wherein first target molecule is a member of a cellular degradation pathway.

11. A chimeric protein according to statement 10 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

12. A chimeric protein according to statement 11 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1-Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

13. A chimeric protein according to statement 10 wherein the cellular degradation pathway is cell mediated autophagy and optionally the peptide ligand is a heat shock cognate of 70kDa (Hsc70) peptide ligand.

14. A chimeric protein according to statement 10 wherein the cellular degradation pathway is lysosomal degradation and the peptide ligand is ALIX, AP-1 , or AP-2.

15. A chimeric protein according to any one of statements 9 to 14 comprising a peptide ligand having an amino acid sequence set out in Table 3.

16. A chimeric protein according to statement wherein the second target molecule is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or

bromodomain protein.

17. A chimeric protein according to statement 16 wherein the second target molecule is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a-synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, BED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

18. A chimeric protein according to any one of statements 16 to 17 comprising a peptide ligand having an amino acid sequence set out in Table 2.

19. A chimeric protein according to any one of the preceding statements comprising a first peptide ligand in the disordered region that binds a target molecule and a second peptide ligand in the coiled-coil domain that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

20. A chimeric protein according to any one of statements 1 to 18 comprising a first peptide ligand in the disordered region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the coiled-coil domain that binds a target molecule.

21. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

22. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 21.

23. An expression vector comprising a nucleic acid according to statement 22.

24. A host cell comprising an expression vector according to statement 23.

25. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 22 to produce the chimeric protein.

26. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a Fibritin scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 21 ; and expressing said chimeric nucleic acid to produce the chimeric protein. 27. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Fibritin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in the coiled-coil domain and the disordered region of the Fibritin scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

28. A method according to statement 27 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

29. A method according to statement 28 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

30. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a Fibritin scaffold, and

(ii) a first peptide ligand located in the coiled-coil subdomain of the Fibritin scaffold of the chimeric protein, and

(iii) a second peptide ligand located in the disordered region of the Fibritin scaffold of the chimeric protein,

wherein at least one amino acid residue in one of the first or second peptide ligand in said library is diverse.

31. A library according to statement 30 wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in the coiled-coil subdomain and the disordered region of the Fibritin scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

32. A library according to statement 31 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

33. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Fibritin scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the disordered region and (b) the coiled-coil subdomain, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in the other of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold.

34. A library according to any one of statements 30-33 wherein the chimeric proteins are according to any one of statements 1 to 21.

35. A library according to any one of statements 30-34 wherein the library is displayed on the surface of particles

36. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Fibritin scaffold,

(ii) a peptide ligand located in one of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold, and;

(iii) a peptide ligand located in the other of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

37. A method of screening a library comprising; (a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Fibritin scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in the disordered region and the coiled-coil subdomain of the Fibritin scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

38. A method according to statement 37 wherein the member of a protein degradation pathway is an E3 ubiquitin ligase.

39. A population of nucleic acids encoding a library according to any one of statements 30-35.

40. A method of producing a library comprising expressing a population of nucleic acids according to statement 39.

41. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Fibritin scaffold,

(ii) a peptide ligand located in one of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold, and;

(iii) a peptide ligand located in another of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins. 42. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 21 , a nucleic acid according to statement 22 or a vector according to statement 23 and a pharmaceutically acceptable excipient.

43. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 21 , a nucleic acid according to statement 22 or a vector according to statement 23 with a pharmaceutically acceptable excipient.

44. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 21 , a nucleic acid according to statement 22 or a vector according to statement 23.

45. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 21 , a nucleic acid according to statement 22 or a vector according to statement 23 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

46. A chimeric protein according to any one of statements 1 to 21 , a nucleic acid according to statement 22 or a vector according to statement 23 for use in a method of diagnosis or treatment in human or animal subject.

47. A chimeric protein according to any one of statements 1 to 21 that binds to a target molecule, a nucleic acid according to statement 22 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 23 for use in the treatment of a disorder associated with the target molecule.

48. Use of a chimeric protein according to any one of statements 1 to 21 that binds to a target molecule, a nucleic acid according to statement 22 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 23 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

49. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 21 that binds to a target molecule, a nucleic acid according to statement 22 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 23 comprising said nucleic acid to an individual in need thereof.

50. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 46 to 49 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(x) aPP scaffold

The following numbered statements relating to the tenth aspect of the invention form part of the description

1. A chimeric protein comprising;

(i) an avian pancreatic polypeptide (aPP) scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or both of the loop and the helical region of the aPP scaffold of the chimeric protein.

2. A chimeric protein according to statement 1 wherein the loop is at a position corresponding to residues 9 to 13 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415.

3. A chimeric protein according to statement 1 wherein the helical region is at a position corresponding to residues 14 to 32 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO:

415.

4. A chimeric protein according to any one of the preceding statements wherein the aPP scaffold comprises the amino acid sequence of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415, an amino acid sequence set out in Table 42, 43 or 44 or a variant of any one of these.

5. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the loop of the aPP scaffold.

6. A chimeric protein according to any one of the preceding statements wherein the peptide ligand is connected to the loop by one or more additional residues.

7. A chimeric protein according to statement 6 wherein the peptide ligand is connected to the loops by a linker.

8. A chimeric protein according to any one of the preceding statements wherein the one or more peptide ligands in the loops are non-hydrophobic.

9. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the helical region of the aPP scaffold.

10. A chimeric protein according to statement 9 wherein the peptide ligand in the helical of the aPP scaffold comprises the amino acid sequence of SEQ ID NO: 417 or a fragment of the amino acid sequence of SEQ ID NO: 417 or a variant of any of these.

11. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the loop and a peptide ligand in the helical region of the aPP scaffold.

12. A chimeric protein according to any one of statements 1 to 11 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

13. A chimeric protein according to any one of statements 1 to 10 comprising a peptide ligand in the loop, and the aPP scaffold binds to a target molecule.

14. A chimeric protein according to any one of statements 1 to 10 comprising a peptide ligand in the helical region, and the aPP scaffold binds to a target molecule.

15. A chimeric protein according to statement 13 or statement 14 comprising a peptide ligand that binds a first target molecule and an aPP scaffold that binds a second target molecule.

16. A chimeric protein according to statement 15 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

17. A chimeric protein according to statement 16 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

18. A chimeric protein according to statement 17 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

19. A chimeric protein according to statement 16 wherein the cellular degradation pathway is cell mediated autophagy and optionally the peptide ligand is a heat shock cognate of 70kDa (Hsc70) peptide ligand.

20. A chimeric protein according to statement 16 wherein the cellular degradation pathway is lysosomal degradation and optionally the peptide ligand is ALIX, AP-1 , or A P-2.

21. A chimeric protein according to statement wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

22. A chimeric protein according to any one of statements 15 to 21 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

23. A chimeric protein according to statement 22 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

24. A chimeric protein according to statement 22 or 23 comprising a peptide ligand having an amino acid sequence set out in Table 1.

25. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the loop that binds a target molecule and a second peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

26. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the helical region that binds a target molecule.

27. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and an aPP scaffold that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 24 comprising an aPP scaffold that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a peptide ligand in the helical region that binds a target molecule.

29. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand in the loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and an aPP scaffold that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 24 comprising a first peptide ligand in the loop that binds a target molecule and an aPP scaffold that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

32. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 31.

33. An expression vector comprising a nucleic acid according to statement 32.

34. A host cell comprising an expression vector according to statement 33.

35. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 32 to produce the chimeric protein.

36. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding an aPP scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 31 ; and expressing said chimeric nucleic acid to produce the chimeric protein.

37. A method according to statement 36 further comprising inserting a second nucleic acid encoding a second peptide ligand into the second nucleic acid encoding an aPP scaffold.

38. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an aPP scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein said peptide ligands are located in (a) the loop and (b) the helical region of the aPP scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

39. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an aPP scaffold that binds a first target molecule; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said peptide ligand,

wherein said peptide ligand is located in (a) the loop or (b) the helical region of the aPP scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein.

40. A method according to statement 38 wherein one of the first or second target molecules is a member of a cellular degradation pathway, optionally an E3 ubiquitin ligase.

41. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) an aPP scaffold,

(ii) two peptide ligands, said peptide ligands being located in (a) the loop and (b) the helical region the aPP scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

42. A library according to statement 41 wherein each chimeric protein in the library comprises (i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in (a) the loop or (b) the helical region of the aPP scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

43. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) an aPP scaffold that binds to a target molecule or a member of a protein degradation pathway,

(ii) a peptide ligand, said peptide ligand being located in (a) the loop or (b) the helical region the aPP scaffold of the chimeric protein,

wherein at least one amino acid residue in the peptide ligand in said library is diverse.

44. A library according to statement 43 wherein each chimeric protein in the library comprises

(i) a peptide ligand for a member of a protein degradation pathway and

(ii) an aPP scaffold for a target molecule,

said peptide ligand being located in (a) the loop or (b) the helical region of the aPP scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different peptide ligands for different members of the protein degradation pathway.

45. A library according to statement 43 wherein each chimeric protein in the library comprises

(i) a peptide ligand for a target molecule and

(ii) an aPP scaffold for a member of a protein degradation pathway,

said peptide ligand being located in (a) the loop or (b) the helical region of the aPP scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different peptide ligands for the target molecule.

46. A library according to any one of statements 42-45 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

47. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an aPP scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold.

48. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an aPP scaffold comprising at least one diverse amino acid residue, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold, and

the aPP scaffold in the chimeric proteins in the second sub-library binds to a second target molecule.

49. A library according to any one of statements 41 to 48 wherein the chimeric proteins are according to any one of statements 1 to 31.

50. A library according to any one of statements 41 to 49 wherein the library is displayed on the surface of particles

51. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an aPP scaffold,

(ii) a peptide ligand located in one of (a) the first loop and (b) the helical region of the aPP scaffold, and;

(iii) a peptide ligand located in another of (a) the first loop and (b) the helical region of the aPP scaffold, wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

52. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an aPP scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in the loop and the helical region of the aPP scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an aPP scaffold that binds to a target molecule,

(ii) a peptide ligand located (a) the first loop and (b) the helical region of the aPP scaffold, and;

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity. 54. A population of nucleic acids encoding a library according to any one of statements 41 to 50.

55. A method of producing a library comprising expressing a population of nucleic acids according to statement 54.

56. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an aPP scaffold,

(ii) a peptide ligand located in one of (a) the loop and (b) the helical region of the aPP scaffold, and;

(iii) a peptide ligand located in the other of (a) the loop and (b) the helical region of the aPP scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an aPP scaffold that binds to a target molecule,

(ii) a peptide ligand located in one of (a) the loop and (b) the helical region of the aPP scaffold, and;

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 with a pharmaceutically acceptable excipient. 60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 31 , a nucleic acid according to statement 32 or a vector according to statement 33 for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 31 that binds to a target molecule, a nucleic acid according to statement 32 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 33 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 63 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(xi) fibronectin scaffold

The following numbered statements relating to the eleventh aspect of the invention form part of the description. 1. A chimeric protein comprising;

(i) a fibronectin (FN3) scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 14 to 15 of SEQ ID NO: 418 or SEQ ID NO: 420.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 25 to 26 of SEQ ID NO: 418 or SEQ ID NO: 420.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 43 to 44 of SEQ ID NO: 418 or SEQ ID NO: 420.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residues 81 to 82 of SEQ ID NO: 418 or SEQ ID NO: 420.

6. A chimeric protein according to any preceding statement wherein the fibronectin scaffold comprises the amino acid sequence of SEQ ID NO: 418 or SEQ ID NO: 420, an amino acid sequence set out in Table 46, an FN3 domain of a sequence set out in Table 47 or a variant of any one of these.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the second loop and a second peptide ligand in the third loop of the fibronectin scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the fourth loop of the fibronectin scaffold.

12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the fibronectin scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the fibronectin scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the fourth loop of the fibronectin scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the fourth loop of the fibronectin scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2. 22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 1.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a fibronectin scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a fibronectin scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a fibronectin scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a fibronectin scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the fibronectin scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the fibronectin scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a fibronectin scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the fibronectin scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third and fourth loops of the fibronectin scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a fibronectin scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a fibronectin scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the fibronectin scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the fibronectin scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is

encapsulated in a liposome.

(xii) Zn finger scaffold

The following numbered statements relating to the twelfth aspect of the invention form part of the description

1. A chimeric protein comprising;

(i) a Zn finger scaffold, and

(ii) one or more peptide ligands, said peptide ligands being located at one or more of the first loop, second loop, and helical region of the Zn finger scaffold of the chimeric protein.

2. A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 6 to 9 of SEQ ID NO: 423.

3. A chimeric protein according to statement 1 or statement 2 wherein the second loop is at a position corresponding to residues 11 to 12 of SEQ ID NO: 423.

4. A chimeric protein according to any one of statements 1 to 3 wherein the helical region is at a position corresponding to 17 to 28 of SEQ ID NO: 423.

5. A chimeric protein according to any one of the preceding statements wherein the Zn finger scaffold comprises the amino acid sequence of SEQ ID NO: 423, or an amino acid sequence set out in Table 49 or Table 50 or a variant of any one of these.

6. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the first loop of the Zn finger scaffold.

7. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the second loop of the Zn finger scaffold

8. A chimeric protein according to any one of statements 6 and 7 wherein the peptide ligand is connected to the loop by one or more additional residues.

9. A chimeric protein according to statement 8 wherein the peptide ligands are connected to the loops by a linker.

10. A chimeric protein according to any one of statements 6 to 9 the peptide ligand is connected to the loop is non-hydrophobic.

11. A chimeric protein according to any one of statements 6 to 10 the peptide ligand in the loop comprises 6 to 10 amino acids.

12. A chimeric protein according to any one of the preceding statements comprising a peptide ligand in the helical region of the Zn finger scaffold.

13. A chimeric protein according to statement 13 wherein the helical region is at a position corresponding to residues 17 to 28 of SEQ ID NO: 423.

14. A chimeric protein according to statement 12 or statement 13 wherein the peptide ligand in the helical region comprises an amino acid sequence of SEQ ID NO: 426 or fragment or variant thereof.

15. A chimeric protein according to any one of statements 12 to 14 comprising a peptide ligand in the first loop of the Zn finger scaffold and a peptide ligand in the helical region of the Zn finger scaffold.

16. A chimeric protein according to any one of statements 12 to 14 comprising a peptide ligand in the second loop of the Zn finger scaffold and a peptide ligand in the helical region of the Zn finger scaffold.

17. A chimeric protein according to any one of statements 1 to 16 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

18. A chimeric protein according to statement 17 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

19. A chimeric protein according to statement 18 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

20. A chimeric protein according to statement 19 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

21. A chimeric protein according to statement 18 wherein the cellular degradation pathway is cell mediated autophagy.

22. A chimeric protein according to statement 21 wherein the peptide ligand is a heat shock cognate of 70kDa (Hsc70) peptide ligand.

23. A chimeric protein according to statement 18 wherein the cellular degradation pathway is lysosomal degradation.

24. A chimeric protein according to statement 23 wherein the peptide ligand is a ligand for ALIX, AP-1 , or AP-2. 25. A chimeric protein according to any one of statements 18 to 24 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

26. A chimeric protein according to any one of statements 18 to 25 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

27. A chimeric protein according to statement 26 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

28. A chimeric protein according to statement 26 or 27 comprising a peptide ligand having an amino acid sequence set out in Table 1.

29. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

30. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

31. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

32. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

33. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the first loop that binds a target molecule.

34. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

35. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

36. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule and a second peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

37. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule, and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

38. A chimeric protein according to any one of statements 20 to 28 comprising a first peptide ligand in the helical region that binds a target molecule, and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

39. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label. 40. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 39.

41. An expression vector comprising a nucleic acid according to statement 40.

42. A host cell comprising an expression vector according to statement 41.

43. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 41 to produce the chimeric protein.

44. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a first peptide ligand into a second nucleic acid encoding a Zn finger scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 39; and expressing said chimeric nucleic acid to produce the chimeric protein.

45. A method according to statement 44 further comprising inserting a second nucleic acid encoding a second peptide ligand into the second nucleic acid encoding a Zn finger scaffold.

46. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a Zn finger scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

47. A method according to statement 46 wherein one of the first or second target molecules is a member of a cellular degradation pathway. 48. A method according to statement 47 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

49. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a Zn finger scaffold,

(ii) two or more peptide ligands, wherein a first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and a second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

50. A library according to statement 49 wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

51. A library according to statement 50 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

52. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a Zn finger scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first loop, second loop and helical region of the Zn finger scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first loop, second loop and helical region of the Zn finger scaffold.

53. A library according to any one of statements 49 to 52 wherein the chimeric proteins are according to any one of statements 1 to 39.

54. A library according to any one of statements 49 to 53 wherein the library is displayed on the surface of particles

55. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a Zn finger scaffold,

(ii) a first peptide ligand located in one of the first loop, second loop and helical region of the Zn finger scaffold, and;

(iii) a second peptide ligand located in another of the first loop, second loop and helical region of the Zn finger scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

56. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a Zn finger scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule,

wherein first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and

(ii) causing degradation of the target molecule in a cell through the protein degradation pathway; and

(c) identifying one or more chimeric proteins in the library which display the activity.

57. A population of nucleic acids encoding a library according to any one of statements 49 to 54.

58. A method of producing a library comprising expressing a population of nucleic acids according to statement 57.

59. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a Zn finger scaffold,

(ii) a first peptide ligand located in one of the first loop, second loop and helical region of the Zn finger scaffold, and;

(iii) a second peptide ligand located in another of the first loop, second loop and helical region of the Zn finger scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

60. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 and a pharmaceutically acceptable excipient.

61. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 with a pharmaceutically acceptable excipient.

62. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41. 63. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

64. A chimeric protein according to any one of statements 1 to 39, a nucleic acid according to statement 40 or a vector according to statement 41 for use in a method of diagnosis or treatment in human or animal subject.

65. A chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 for use in the treatment of a disorder associated with the target molecule.

66. Use of a chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

67. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 39 that binds to a target molecule, a nucleic acid according to statement 40 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 41 comprising said nucleic acid to an individual in need thereof.

68. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 64 to 67 wherein the chimeric protein, nucleic acid or vector is encapsulated in a liposome.

(xiii) SH3 scaffold.

The following numbered statements relating to the thirteenth aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) an SH3 scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein. 2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 9 to 24 of SEQ ID NO: 653; residues 24 to 39 of SEQ ID NO: 655; or 13 to 21 of SEQ ID NO: 657.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 31 to 35 of SEQ ID NO: 653; residues 45 to 56 of SEQ ID NO: 655; or residues 32 to 41 of SEQ ID NO: 657.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 44 to 46 of SEQ ID NO: 653 residues 62 to 63 of SEQ ID NO: 655; or residues 53 to 62 of SEQ ID NO: 657.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residues 55 to 56 of SEQ ID NO: 653; residues 69 to 71 of SEQ ID NO: 655; or residues 68 to 70 of SEQ ID NO: 657.

6. A chimeric protein according to any preceding statement wherein the SH3 scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 427 to 652, 653, 655 and 657 or a variant thereof.

7. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

8. A chimeric protein according to statement 7 wherein the peptide ligands are connected to the loops by a linker.

9. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

10. A chimeric protein according to any preceding statement comprising a first peptide ligand in the first loop and a second peptide ligand in the second loop of the SH3 scaffold.

11. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the third loop of the SH3 scaffold. 12. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the first loop and a second peptide ligand in the fourth loop of the SH3 scaffold.

13. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the third loop of the SH3 scaffold.

14. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the second loop and a second peptide ligand in the fourth loop of the SH3 scaffold

15. A chimeric protein according to any one of statements 1 to 9 comprising a first peptide ligand in the third loop and a second peptide ligand in the fourth loop of the SH3 scaffold

16. A chimeric protein according to any one of statements 1 to 15 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

17. A chimeric protein according to statement 16 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

18. A chimeric protein according to statement 17 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

19. A chimeric protein according to statement 18 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP.

20. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

21. A chimeric protein according to statement 17 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

22. A chimeric protein according to any one of statements 17 to 21 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

23. A chimeric protein according to any one of statements 17 to 22 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

24. A chimeric protein according to statement 23 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, a- synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

25. A chimeric protein according to any one of statements 16 to 24 comprising a peptide ligand having an amino acid sequence set out in Table 1.

26. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

27. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule.

28. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

30. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the first loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule.

32. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

34. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

36. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fourth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 37. A chimeric protein according to any one of statements 16 to 25 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fourth loop that binds a target molecule

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding an SH3 scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding an SH3 scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein 45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) an SH3 scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) an SH3 scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the SH3 scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the SH3 scaffold.

51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) an SH3 scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the SH3 scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third and fourth loops of the SH3 scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) an SH3 scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule, said first and second peptide ligands being located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) an SH3 scaffold,

(ii) a first peptide ligand located in one of the first, second, third and fourth loops of the SH3 scaffold;

(iii) a second peptide ligand located in another of one of the first, second, third and fourth loops of the SH3 scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector.

62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is encapsulated in a liposome.

(xiv) Cystine knot Scaffolds

The following numbered statements relating to the fourtheenth aspect of the invention form part of the description.

1. A chimeric protein comprising;

(i) a Cystine Knot (CK) scaffold, and

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third, fourth, fifth and sixth loops of the CK scaffold of the chimeric protein.

2 A chimeric protein according to statement 1 wherein the first loop is at a position corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842, residues 2 to 7 of SEQ ID NO: 844; residues 3 to 8 of SEQ ID NO: 846 and residues 3 to 4 of SEQ ID NO: 848.

3 A chimeric protein according to any preceding statement wherein the second loop is at a position corresponding to residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 842, residues 9 to 13 of SEQ ID NO: 844; residues 10 to 14 of SEQ ID NO: 846 and residues 6 to 15 of SEQ ID NO: 848.

4. A chimeric protein according to any preceding statement wherein the third loop is at a position corresponding to residues 1 1 to 14 of SEQ ID NO: 840; residues 1 1 to 16 of SEQ ID NO: 842, residues 15 to 17 of SEQ ID NO: 844; residues 16 to 18 of SEQ ID NO: 846 and residues 17 to 19 of SEQ ID NO: 848.

5. A chimeric protein according to any preceding statement wherein the fourth loop is at a position corresponding to residue 16 of SEQ ID NO: 840; residue 18 of SEQ ID NO: 842, residue 19 of SEQ ID NO: 844; residue 20 of SEQ ID NO: 846 and residues 21 to 27 of SEQ ID NO: 848.

6. A chimeric protein according to any preceding statement wherein the fifth loop is at a position corresponding to residues 18 to 21 of SEQ ID NO: 840; residues 20 to 23 of SEQ ID NO: 842, residues 21 to 25 of SEQ ID NO: 844; residues 22 to 26 of SEQ ID NO: 846 and residues 29 to 32 of SEQ ID NO: 848.

7. A chimeric protein according to any preceding statement wherein the sixth loop is at a position corresponding to residues 23 to 30 of SEQ ID NO: 840; residues 25 to 31 of SEQ ID NO: 842, residues 27 to 35 of SEQ ID NO: 844; residues 28 to 30 of SEQ ID NO: 846 and residue 34 of SEQ ID NO: 848.

8. A chimeric protein according to any preceding statement wherein the CK scaffold comprises an amino acid sequence of any one of SEQ ID NOs: 1 , 3, 5, 7 and 9 or a variant of any one of these.

9. A chimeric protein according to any preceding statement wherein the peptide ligands are connected to the loops by one or more additional residues.

10. A chimeric protein according to statement 9 wherein the peptide ligands are connected to the loops by a linker. 11. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-hydrophobic.

12. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops are non-helical.

13. A chimeric protein according to any preceding statement wherein the one or more peptide ligands in the loops comprise 3 to 50 amino acids.

14. A chimeric protein according to any preceding statement comprising a first peptide ligand in the second loop and a second peptide ligand in the sixth loop of the CK scaffold.

15. A chimeric protein according to any one of statements 1 to 13 comprising a first peptide ligand in the third loop and a second peptide ligand in the sixth loop of the CK scaffold.

16. A chimeric protein according to any one of statements 1 to 13 comprising a first peptide ligand in the fifth loop and a second peptide ligand in the sixth loop of the CK scaffold.

17. A chimeric protein according to any one of statements 1 to 13 comprising a first peptide ligand in the third loop and a second peptide ligand in the fifth loop of the CK scaffold.

18. A chimeric protein according to any one of statements 1 to 17 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.

19. A chimeric protein according to statement 18 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

20. A chimeric protein according to statement 19 wherein the member of a cellular degradation pathway is an E3 ubiquitin ligase.

21. A chimeric protein according to statement 20 wherein the E3 ubiquitin ligase is Mdm2, SCF ^Skp2 , Cul3-Keap1 , Cul3-SPOP, APC/C, SIAH, SCF ^Fbw7 , SCF ^Fbw8 , Cul4-DDB1- Cdt2, DDB1-CUL4, SOCS box-Cul5-SPSB4, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21 , Nedd4, Elongin C or b-TRCP. 22. A chimeric protein according to statement 19 wherein the member of the cellular degradation pathway is heat shock cognate of 70kDa (Hsc70).

23. A chimeric protein according to statement 19 wherein the member of the cellular degradation pathway is ALIX, AP-1 , or AP-2.

24. A chimeric protein according to any one of statements 19 to 23 wherein the peptide ligand for the member of the cellular degradation pathway comprises an amino acid sequence set out in Table 2.

25. A chimeric protein according to any one of statements 19 to 24 wherein the other of the first or second target molecules is a receptor, enzyme, antigen, polynucleotide, oligosaccharide, integral membrane protein, G protein coupled receptor (GPCR), transcription factor, transcriptional regulator or bromodomain protein.

26. A chimeric protein according to statement 25 wherein the other of the first or second target molecules is b-catenin, KRAS, tan ky rase, c-myc, n-myc, ras, notch and aurora A, o synuclein, b-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, oncogenic histone methyltransferase, EWS-FLI1 (Ewing’s sarcoma-friend leukemia integration 1), TEL-AML1 , TAL1 (T-cell acute lymphocytic leukemia protein 1), PP2A; BRD4, PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1), WDR5, CdK2, PP2A, EED, MCL1 , GSK3, CtBP, Bcl9, Jun, Fos, Tau, RTK, GHR, PD-L1 , and BCR (breakpoint cluster region)-ABL.

27. A chimeric protein according to any one of statements 18 to 26 comprising a peptide ligand having an amino acid sequence set out in Table 1.

28. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the sixth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

29. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the sixth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the second loop that binds a target molecule. 30. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the sixth loop that binds a target molecule and a second peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

31. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the sixth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the third loop that binds a target molecule.

32. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the fifth loop that binds a target molecule and a second peptide ligand in the sixth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

33. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the fifth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the sixth loop that binds a target molecule.

34. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the third loop that binds a target molecule and a second peptide ligand in the fifth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase.

35. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the third loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fifth loop that binds a target molecule.

36. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the second loop that binds a target molecule and a second peptide ligand in the fifth loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase. 37. A chimeric protein according to any one of statements 18 to 27 comprising a first peptide ligand in the second loop that binds a member of a cellular degradation pathway, e.g. an E3 ubiquitin ligase, and a second peptide ligand in the fifth loop that binds a target molecule.

38. A chimeric protein according to any one of the preceding statements further comprising a targeting domain, intracellular transfer domain, stabilising domain,

oligomerisation domain, cytotoxic agent, therapeutic agent and/or a detectable label.

39. A nucleic acid encoding a chimeric protein according to any one of statements 1 to 38.

40. An expression vector comprising a nucleic acid according to statement 39.

41. A host cell comprising an expression vector according to statement 40.

42. A method of producing a chimeric protein comprising expressing a nucleic acid according to statement 39 to produce the chimeric protein.

43. A method of producing a chimeric protein comprising;

inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a CK scaffold to produce a chimeric nucleic acid encoding a chimeric protein according to any one of statements 1 to 38; and expressing said chimeric nucleic acid to produce the chimeric protein.

44. A method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;

providing a nucleic acid encoding a CK scaffold; and

incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third, fourth fifth and sixth loops of the CK scaffold of the chimeric protein; and

expressing the nucleic acid to produce said protein

45. A method according to statement 44 wherein one of the first or second target molecules is a member of a cellular degradation pathway.

46. A method according to statement 44 or 45 wherein one of the first or second target molecules is an E3 ubiquitin ligase.

47. A library comprising chimeric proteins, each chimeric protein in the library

comprising;

(i) a CK scaffold,

(ii) two or more peptide ligands, said peptide ligands being located in two or more of the first, second, third, fourth fifth and sixth loops of the CK scaffold of the chimeric protein, wherein at least one amino acid residue in a peptide ligand in said library is diverse.

48. A library according to statement wherein each chimeric protein in the library comprises

(i) a first peptide ligand for a member of a protein degradation pathway and

(ii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third, fourth fifth and sixth loops of the CK scaffold of the chimeric protein,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins in the library comprising different combinations of said first and second peptide ligands.

49. A library according to statement 48 wherein the member of the cellular degradation pathway is an E3 ubiquitin kinase.

50. A library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

(i) a CK scaffold, and

(ii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third, fourth fifth and sixth loops of the CK scaffold, and

the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third, fourth fifth and sixth loops of the CK scaffold. 51. A library according to any one of statements 47-50 wherein the chimeric proteins are according to any one of statements 1 to 38.

52. A library according to any one of statements 47-51 wherein the library is displayed on the surface of particles

53. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising;

(i) a CK scaffold,

(ii) a first peptide ligand located in one of the first, second, third, fourth fifth and sixth loops of the CK scaffold, and;

(iii) a second peptide ligand located in another of the first, second, third, fourth fifth and sixth loops of the CK scaffold,

wherein at least one amino acid residue in the peptide ligands in said library is diverse,

(b) screening the library for chimeric proteins which display a binding activity, and

(c) identifying one or more chimeric proteins in the library which display the binding activity.

54. A method of screening a library comprising;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a CK scaffold;

(ii) a first peptide ligand for a member of a cellular degradation pathway and

(iii) a second peptide ligand for a target molecule,

said first and second peptide ligands being located in two of the first, second, third, fourth fifth and sixth loops of the CK scaffold of the chimeric protein,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity. 55. A population of nucleic acids encoding a library according to any one of statements 47 to 52.

56. A method of producing a library comprising expressing a population of nucleic acids according to statement 55.

57. A method of producing a library of chimeric proteins comprising;

(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising

(i) a CK scaffold,

(ii) a first peptide ligand located in one of first, second, third, fourth fifth and sixth loops of the CK scaffold;

(iii) a second peptide ligand located in another of the first, second, third, fourth fifth and sixth loops of the CK scaffold,

wherein the peptide ligands in said population are diverse, and

(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

58. A pharmaceutical composition comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 and a pharmaceutically acceptable excipient.

59. A method of producing a pharmaceutical composition comprising formulating a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a pharmaceutically acceptable excipient.

60. A population of liposomes comprising a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40.

61. A method of producing a population of liposomes comprising admixing a chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 with a lipid solution and evaporating said solution to produce liposomes encapsulating said chimeric protein, nucleic acid or vector. 62. A chimeric protein according to any one of statements 1 to 38, a nucleic acid according to statement 39 or a vector according to statement 40 according to statement for use in a method of diagnosis or treatment in human or animal subject.

63. A chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 for use in the treatment of a disorder associated with the target molecule.

64. Use of a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 in the manufacture of a medicament for use in the treatment of a disorder associated with the target molecule.

65. A method of treatment of a disorder associated with the target molecule comprising; administering a chimeric protein according to any one of statements 1 to 38 that binds to a target molecule, a nucleic acid according to statement 39 that encodes a chimeric protein that binds to a target molecule or a vector according to statement 40 comprising said nucleic acid to an individual in need thereof.

66. A chimeric protein, nucleic acid or vector for use, use or method according to any one of statements 62 to 65 wherein the chimeric protein, nucleic acid or vector is encapsulated in a liposome.