SPYCATCHER/SPYTAG CYCLISED PEPTIDES DISPLAYED ON

BACTERIOPHAGE

Technical Field

The present disclosure generally relates to binding proteins and protein binding partners for display of peptides and various uses thereof.

Background

Peptides are attractive diagnostic and therapeutic targets due to their varied physiological roles and potentially high potency and target specificity. However, the identification and development of such peptides remains challenging.

Peptides sometimes bind to their targets with modest affinity due to entropic thermodynamic reasons because the exhibit insufficient conformational constraint. While constraint may be conferred through cyclisation of the peptides, but this can involve complex chemistries which may not be feasible for the construction of large libraries.

Many peptides when expressed in a host cell are susceptible to degradation by proteases and peptidases. This is normally because these peptides are unable to form stable tertiary structures. While one or more disulfide bonds may be added to increase the stability of certain peptides, disulfide bonds are susceptible to reduction in certain extracellular environments such as in blood and within cells.

Moreover, a significant proportion of peptides that are stably expressed accumulate in insoluble aggregates. Peptides that are present in inclusion bodies may be misfolded, inactive and/or denatured. The process of obtaining bioactive peptides from inclusion bodies requires extensive processing comprising isolation, solubilization, refolding and purification.

Peptides can sometimes be used as receptor binding domains (RBD) for targeting specific cell types and/or concentrating drugs to diseased tissues such as tumours. However, such peptide-derived RBDs are often unstable and can be challenging to conjugate to their payloads.

In the in vivo diagnostics and drug conjugate fields, there exists a desirability to combine a protein based functionality (such as receptor targeting, or an effect on a biological target) with a synthetic peptide component which can be used as a 'handle' to attach a moiety such as an imaging probe or a small molecule toxin molecule, respectively. Accordingly, there remains an unmet need for a system for displaying a wide range of constrained peptides and methods for screening constrained peptide libraries useful in biological and therapeutic applications. Summary

The present disclosure provides reagents and methods for displaying peptides, e.g., in a constrained form. Such reagents and methods can provide for additional stability of a peptide. The reagents and methods of the disclosure additionally permit modular production of proteins and complexes displaying a plurality of peptides. For example, a protein or complex of the disclosure may display a peptide capable of inhibiting an intracellular protein interaction and a cell penetrating peptide to allow for intracellular delivery. Similarly, a protein or complex of the disclosure may display a therapeutic agent or peptide and a peptide that is a receptor binding domain to allow for cell targeting and concentration on a disease tissue or cell of interest. Likewise a protein or complex of the disclosure could be used for diagnostic or theranostic purposes. The present disclosure also provides reagents and methods to facilitate screening for peptides having various biological activities.

The present disclosure provides a complex comprising:

a) a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide; and

c) a protein binding partner;

wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond, the binding protein is linked a first terminus of the peptide and the peptide tag is linked to a second terminus of the peptide.

In one example, the complex comprises:

a) a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide; and

c) a protein binding partner;

wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond, the binding protein is linked the N' terminus of the peptide and the peptide tag is linked to the C terminus of the peptide.

In another example, the complex comprises:

a) a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide; and

c) a protein binding partner;

wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond, the binding protein is linked the C terminus of the peptide and the peptide tag is linked to the N' terminus of the peptide.

In one example, the binding protein disclosed herein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 2. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 3. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 4. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 5. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 6. For example , the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 7. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 8. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 9. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 10. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 11. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 12. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 13. For example, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 14.

In an exemplary form of the present disclosure, the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 1.

In one example, the protein binding partner described herein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

In another example, the protein binding partner described herein comprises an amino acid set forth in SEQ ID NO: 15. For example, the protein binding partner described comprises an amino acid set forth in SEQ ID NO: 16. For example, the protein binding partner comprises an amino acid set forth in SEQ ID NO: 17. For example, the protein binding partner comprises an amino acid set forth in SEQ ID NO: 18. In one example, the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein is an autocatalytic reaction.

In one example, the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein occurs within at least 24 hours of contacting a binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 12 hours of contacting a binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 6 hours of contacting a binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 3 hours of contacting a binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 2 hours of contacting a binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 1 hour of contacting a binding protein with a protein binding partner.

In one example, the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein is stable under conditions that may dissociate non-covalently linked proteins. For example, the intermolecular isopeptide bond is stable at 80°C. For example, the intermolecular isopeptide bond is stable at 85°C. For example, the intermolecular isopeptide bond is stable at 90°C. For example, the intermolecular isopeptide bond is stable at 95 °C. For example, the intermolecular isopeptide bond is stable at 100°C.

In one example, any binding protein or protein binding partner described herein is linked to a detectable label. In one example, the detectable label is a fluorescent tag. In another example, the detectable label is a chemical tag. In another example, the detectable label is an epitope tag. In another example, the detectable label is a fluorescent tag. In another example, the detectable label is an isobaric tag. In another example, the detectable label is a radiochemical tag. In another example, the detectable label is a fluorescent tag. In another example, the detectable label is a biotin. In another example, the detectable label is a fluorescent tag. In another example, the detectable label is an acyl carrier protein tag. In another example, the detectable label is a fluorescent tag. In another example, the detectable label is streptavidin.

In one example, any binding protein or protein binding partner described herein is linked to a therapeutic agent.

In one example, any binding protein or protein binding partner described herein is linked to a toxin.

In one example, any binding protein or protein binding partner described herein is linked to a cell penetrating peptide. In one example, any binding protein or protein binding partner described herein is linked to a RNAi agent. In one example, the RNAi agent is a small interfering RNA. In one example, the RNAi agent is a short hairpin RNA. In one example, the RNAi agent is a microRNA. In one example, any binding protein or protein binding partner described herein is linked to a nucleic acid therapeutic.

In one example, any binding protein or protein binding partner described herein is linked to a short activating RNA.

In one example, any binding protein or protein binding partner described herein is linked to an antigen.

In one example, any complex described herein is displayed on a bacteriophage.

In one example, the bacteriophage is a T phage. In another example, the bacteriophage is a filamentous phage. In another example, the bacteriophage is a lysogenic bacteriophage. In another example, the bacteriophage is a lambda phage.

In another example, any complex described herein is displayed on a nanoparticle or a micro particle.

In one example, any complex described herein is displayed within in a cell. In one example, the cell is a bacterial cell. In another example, the cell is a yeast cell. In another example, the cell is a mammalian cell.

The present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:

i) incubating a displayed complex described herein with a molecule; and ii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide in the complex which is linked at a first end to a binding protein and at a second end to a protein binding partner).

The present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:

i) providing a displayed complex disclosed herein;

ii) incubating the displayed complex of provided in step i) with a molecule; and iii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide in the complex which is linked at a first end to a binding protein and at a second end to a protein binding partner).

In one example, the method additionally comprises a washing step to remove any unbound peptides.

In one example, the method additionally comprises a filtration step to remove any unbound peptides. In one example, the method additionally comprises incubating the displayed complex with a non-target molecule prior to step ii). For example, the non-target molecule is a cell that does not express the molecule.

In one example, the method additionally comprises isolating the peptide that interacts with the molecule.

In one example, detecting the presence of an interaction between the molecule and the peptide comprises detecting a detectable label linked to a binding protein or a protein binding partner. For example, the detectable label is a fluorescent tag. In another example, detecting the presence of an interaction between the molecule and the peptide comprises measuring a refractive index. In another example, detecting the presence of an interaction between the molecule and the peptide comprises measuring an acoustic or optical resonance.

In one example, detecting the presence of an interaction between the molecule and the peptide additionally comprises detecting a peptide that specifically binds to a molecule. For example, detecting the presence of an interaction between the molecule and the peptide comprises a peptide that binds to a molecule with an equilibrium constant (KD) of 100 nM or less.

In one example, the molecule is a cell surface receptor. In one example, the cell surface receptor is epithelial growth factor receptor (EGFR). In another example, the cell surface receptor is C-X-C chemokine receptor type 4 (CXCR4). In another example, the cell surface receptor is folate receptor alpha protein. In another example, the cell surface receptor is a folate receptor beta protein. In yet another example, the cell surface receptor is CD19, CD20 or CD22 receptors expressed on lymphoid cells.

In one example, the molecule is an intracellular receptor.

In one example, the molecule is a transcription factor. In one example, the transcription factor is a STAT family protein. In another example, the transcription factor surface receptor is c-Myc.

In another example, the molecule is a RNA binding protein. In one example, the RNA binding protein is YB-1.

In one example, a molecule described herein is immobilised on a solid support.

The present disclosure also provides a method for identifying a peptide capable of translocating a membrane of a cell comprising:

i) contacting a cell with a displayed complex disclosed herein; and

ii) detecting the peptide in the cell (i.e. the peptide in the complex which is linked at a first end to a binding protein and at a second end to a protein binding partner). In one example, the method additionally comprises incubating the cell and the complex for a time and under conditions sufficient for the complex to enter a cell.

In one example, the method additionally comprises washing the cell to remove any unbound modified binding proteins.

In one example, the method additionally comprises isolating the peptide.

In one example, detecting the peptide in the cell comprises detecting a detectable label linked to the modified binding protein. In one example, the detectable label is a fluorescent tag.

The present disclosure also provides a modified binding protein. Any binding protein described herein may be modified.

In one example, a modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises one or more peptides inserted into one or more regions of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.

In one example, a modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 of SEQ ID NO: 1.

In another example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.

In another example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In another example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 or 48-50 of SEQ ID NO: 1.

In one example, a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 of SEQ ID NO: 1.

In one example, a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.

In one example, a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.

In one example, a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1. In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a (e.g. one) peptide inserted into a region of the binding protein corresponding to amino acid residues amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1 and another peptide (or peptides) is inserted into a region of the binding protein corresponding to amino acid residues amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 27 and 29 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 27 and 30 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 29 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 30 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 31 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 29 and 30 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 29 and 31 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 30 and 31 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48 and 50 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 48 and 49 of SEQ ID NO: 1.

In one example, when the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48 and 50 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 49 and 50 of SEQ ID NO: 1.

In one example, a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

In another example, a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 15.

In another example, a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 16.

In another example, a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 17.

In another example, a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 18.

In one example, the intermolecular isopeptide bond formed between a modified binding protein described herein and a protein binding partner described herein is an autocatalytic reaction.

In one example, the intermolecular isopeptide bond formed between a modified binding protein described herein and protein binding partner described herein occurs within at least 24 hours of contacting the modified binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 12 hours of contacting the modified binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 6 hours of contacting the modified binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 3 hours of contacting the modified binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 2 hours of contacting the modified binding protein with a protein binding partner. For example, the intermolecular isopeptide bond may form within 1 hour of contacting the modified binding protein with a protein binding partner.

In one example, the intermolecular isopeptide bond formed between a modified binding protein described herein and protein binding partner described herein is stable under conditions that may dissociate non-covalently linked proteins. For example, the intermolecular isopeptide bond is stable at 80°C. For example, the intermolecular isopeptide bond is stable at 85°C. For example, the intermolecular isopeptide bond is stable at 90°C. For example, the intermolecular isopeptide bond is stable at 95 °C. For example, the intermolecular isopeptide bond is stable at 100°C.

In one example, a modified binding protein described herein is linked to a detectable label. In one example, a protein binding partner described herein is linked to a detectable label. In one example, the detectable label is a fluorescent tag. In another example, the detectable label is a chemical tag. In another example, the detectable label is an epitope tag. In another example, the detectable label is an isobaric tag. In another example, the detectable label is a radiochemical tag. In another example, the detectable label is a biotin. In another example, the detectable label is an acyl carrier protein tag. In another example, the detectable label is streptavidin.

In one example, a modified binding protein described herein is linked to a therapeutic agent.

In one example, a protein binding partner described herein is linked to a therapeutic agent.

In one example, a modified binding protein described herein is linked to a toxin. In one example, a protein binding partner described herein is linked to a toxin. In one example, a modified binding protein described herein is linked to a small molecule.

In one example, a protein binding partner described herein is linked to a small molecule.

In one example, a modified binding protein described herein is linked to a cell penetrating peptide.

In one example, a modified binding protein described herein is linked to a cell penetrating peptide.

In one example, a modified binding protein described herein is linked to a RNAi agent. In one example, a protein binding partner described herein is linked to a RNAi agent. In one example, the RNAi agent is a small interfering RNA. In one example, the RNAi agent is a short hairpin RNA. In one example, the RNAi agent is a microRNA. In one example, any modified binding protein or protein binding partner described herein is linked to nucleic acid therapeutic.

In one example, a modified binding protein described herein is linked to an antigen.

In one example, a protein binding partner described herein is linked to an antigen.

In one example, a modified binding protein described herein is linked to a short activating RNA.

In one example, a protein binding partner described herein is linked to a short activating RNA.

In one example, a modified binding protein described herein is displayed on a particle.

In one example, a modified binding protein described herein is displayed on a bacteriophage. In one example, the bacteriophage is a T phage. In another example, the bacteriophage is a filamentous phage. In another example, the bacteriophage is a lysogenic bacteriophage. In another example, the bacteriophage is a lambda phage, any modified binding protein described herein is displayed on a nanoparticle or a microparticle.

In one example, a modified binding protein described herein is displayed on the surface of a cell.

In one example, a modified binding protein described herein is displayed on the surface of a bacterial cell.

In one example, a modified binding protein described herein is displayed on the surface of a yeast cell.

In one example, a modified binding protein described herein is displayed within in a cell.

In one example, a modified binding protein described herein is displayed within in a bacterial cell.

In another example, a modified binding protein described herein is displayed within in a yeast cell.

In another example, a modified binding protein described herein is displayed within in a mammalian cell. In one example, a modified binding protein is displayed within in a CHO-K1 cell. In another example, a modified binding protein is displayed within in a HEK293 cell.

The present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising: i) incubating a modified binding protein disclosed herein with a molecule; and ii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide inserted into a region of the binding protein).

The present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:

i) providing a modified binding protein disclosed herein;

ii) incubating the modified binding protein of provided in step i) with a molecule; and

iii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide inserted into a region of the binding protein).

In one example, the method additionally comprises a washing step to remove any unbound modified binding proteins.

In one example, the method additionally comprises a filtration step to remove any unbound modified binding proteins.

In one example, the method additionally comprises incubating the modified binding protein with a non-target molecule prior to step ii). For example, the non-target molecule is a cell that does not express the molecule.

In one example, the method additionally comprises isolating the peptide (i.e. the peptide inserted into a region of the binding protein) that interacts with the molecule.

In one example, detecting the presence of an interaction between the molecule and the peptide comprises detecting a detectable label linked to a binding protein or a protein binding partner. For example, the detectable label is a fluorescent tag. In another example, detecting the presence of an interaction between the molecule and the peptide comprises measuring a refractive index. In another example, detecting the presence of an interaction between the molecule and the peptide comprises measuring an acoustic or optical resonance.

In one example, detecting the presence of an interaction between the molecule and the peptide additionally comprises detecting a peptide that specifically binds to a molecule. For example, detecting the presence of an interaction between the molecule and the peptide comprises a peptide that binds to a molecule with an equilibrium constant (KD) of 100 nM or less.

In one example, the molecule is a cell surface receptor. In one example, the cell surface receptor is epithelial growth factor receptor (EGFR). In another example, the cell surface receptor is C-X-C chemokine receptor type 4 (CXCR4). In another example, the cell surface receptor is folate receptor alpha protein. In another example, the cell surface receptor is a folate receptor beta protein. In one example, the molecule is an intracellular receptor.

In one example, the molecule is a transcription factor is a transcription factor, a transcriptional co-activator or a co-repressor. In one example, the transcription factor is a STAT family protein. In another example, the transcription factor is Myc. In another example, the transcriptional co-activator is beta catenin. In another example, the transcriptional co-activator is Master Mind Like Protein (MAML).

In one example, the intracellular receptor is an RNA binding protein. In one example, the RNA binding protein is YB-1.

In one example, a molecule described herein, is immobilised on a solid support. The present disclosure also provides a method for directing a molecule to a target comprising:

i) contacting a cell with a modified binding protein described herein linked to a molecule;

wherein the cell expresses a target linked to a protein binding partner described herein, and wherein the modified binding protein forms a covalent isopeptide bond with the protein binding partner such that the molecule is directed to the target.

In one example, contacting at step i) is for a time and under conditions sufficient for the modified binding protein linked to a molecule to enter a cell.

In one example, the method additionally comprises determining or identifying that the modified binding protein linked to a molecule has been directed to a target.

In one example, the method additionally comprises producing cells that expresses a target linked to the protein binding partner.

In one example, the molecule is a therapeutic agent.

In one example, the molecule is a toxin.

In one example, the molecule is a transcription factor.

In one example, the molecule is an enzyme.

In one example, the molecule is a RNAi agent. In one example, the RNAi agent is a small interfering RNA. In one example, the RNAi agent is a short hairpin RNA. In one example, the RNAi agent is a microRNA. In one example, the molecule is an antigen. In another example, the molecule is a nucleic acid therapeutic.

In one example, the molecule is a short activating RNA.

In one example, the target is expressed in a subcellular compartment.

In one example, the target is a cell surface receptor.

In one example, the target is an intracellular receptor.

In one example, the target is a transcription factor.

In one example, the target is component of a membrane receptor. In one example, the target is component of a signalling adaptor protein.

The present disclosure also provides a method for identifying a peptide capable of translocating a membrane of a cell comprising:

i) contacting a cell with a modified binding protein disclosed herein; and ii) detecting the peptide in the cell (i.e. the peptide inserted into a region of the binding protein).

In one example, the method additionally comprises incubating the cell and the modified binding protein for a time and under conditions sufficient for the modified binding protein linked to enter a cell.

In one example, the method additionally comprises washing the cell to remove any unbound modified binding proteins.

In one example, the method additionally comprises isolating the peptide.

In one example, detecting the peptide in the cell comprises detecting a detectable label linked to the modified binding protein. In one example, the detectable label is a fluorescent tag.

The present disclosure also provides a method for identifying a receptor binding domain, the method comprising:

i) contacting a cell with a complex comprising:

a modified binding protein disclosed herein; and

a protein binding partner disclosed herein linked to a cell penetrating peptide; wherein the modified binding protein is linked with the protein binding partner by a covalent isopeptide bond, and

ii) detecting the complex within the cell;

wherein the detection of the complex within the cell is indicative that the peptide (i.e. the peptide inserted into a region of the binding protein) is a receptor binding domain.

In one example, the protein binding partner is additionally linked to a molecule. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin.

The present disclosure also provides a method for identifying a receptor binding domain, the method comprising:

i) providing a complex comprising:

a modified binding protein disclosed herein; and

a protein binding partner disclosed herein linked to a cell penetrating peptide; wherein the modified binding protein forms a covalent isopeptide bond with the protein binding partner,

ii) contacting a cell with the complex; and iii) detecting the complex within a cell;

wherein the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a receptor binding domain.

In one example, the protein binding partner is additionally linked to a molecule. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin. In yet another example the molecule is a diagnostic or imaging agent.

The present disclosure also provides a method for identifying a cell penetrating, the method comprising:

i) contacting a cell with a complex comprising:

a modified binding protein disclosed herein; and

a protein binding partner disclosed herein linked to a receptor binding domain;

wherein the modified binding protein is linked with the protein binding partner by a covalent isopeptide bond, and

ii) detecting the complex within a cell;

wherein the detection of a complex within a cell is indicative that the peptide (i.e. the peptide inserted into a region of the binding protein) is a cell penetrating peptide.

The present disclosure also provides a method for identifying a cell penetrating, the method comprising:

i) providing a complex comprising:

a modified binding protein disclosed herein; and

a protein binding partner disclosed herein linked to a receptor binding domain;

wherein the modified binding protein forms a covalent isopeptide bond with the protein binding partner,

ii) contacting a cell with the complex; and

iii) detecting the complex within a cell;

wherein the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.

In one example, the protein binding partner is additionally linked to a molecule. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin.

The present disclosure also provides a method for identifying a cell penetrating, the method comprising: i) contacting a cell with a complex comprising a modified binding protein disclosed herein linked to a receptor binding domain; and

ii) detecting the complex within a cell;

wherein the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.

In one example, the protein binding partner is additionally linked to a molecule. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin.

The present disclosure also provides a method for identifying a cell penetrating, the method comprising:

i) providing a complex comprising a modified binding protein disclosed herein linked to a receptor binding domain;

ii) contacting a cell with the complex; and

iii) detecting the complex within a cell;

wherein the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.

In one example, the protein binding partner is additionally linked to a molecule. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin.

Key to Sequence Listing

SEQ ID NO: 1 Amino acid sequence of a binding ; protein

SEQ ID NO: 2 Amino acid sequence of a binding ; protein

SEQ ID NO: 3 Amino acid sequence of a binding ; protein

SEQ ID NO: 4 Amino acid sequence of a binding ; protein

SEQ ID NO: 5 Amino acid sequence of a binding ; protein

SEQ ID NO: 6 Amino acid sequence of a binding ; protein

SEQ ID NO: 7 Amino acid sequence of a binding ; protein

SEQ ID NO: 8 Amino acid sequence of a binding ; protein

SEQ ID NO: 9 Amino acid sequence of a binding ; protein

SEQ ID NO: 10 Amino acid sequence of a binding ; protein

SEQ ID NO: 11 Amino acid sequence of a binding ; protein

SEQ ID NO: 12 Amino acid sequence of a binding ; protein

SEQ ID NO: 13 Amino acid sequence of a binding ; protein

SEQ ID NO: 14 Amino acid sequence of a binding ; protein

SEQ ID NO: 15 Amino acid sequence of a protein binding partner SEQ ID NO: 16 Amino acid sequence of a protein binding partner

SEQ ID NO: 17 Amino acid sequence of a protein binding partner

SEQ ID NO: 18 Amino acid sequence of a protein binding partner

SEQ ID NO: 19 Amino acid sequence of a mutant protein binding partner

SEQ ID NO: 20 Amino acid sequence of a peptide linker

SEQ ID NO: 21 Amino acid sequence of a peptide linker

SEQ ID NO: 22 Amino acid sequence of a peptide linker

SEQ ID NO: 23 Amino acid sequence of a bovine pancreatic trypsin inhibitor

SEQ ID NO: 24 Amino acid sequence of a mutant bovine pancreatic trypsin inhibitor

SEQ ID NO: 25 Amino acid sequence of a protein binding partner-bovine pancreatic trypsin inhibitor-binding protein cassette

SEQ ID NO: 26 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor-protein binding partner cassette

SEQ ID NO: 27 Amino acid sequence of a binding protein-mutant bovine pancreatic trypsin inhibitor-partner cassette

SEQ ID NO: 28 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor-mutant protein binding partner

SEQ ID NO: 29 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor -mutant protein binding partner

SEQ ID NO: 30 Amino acid sequence of a modified binding protein-mutant bovine pancreatic trypsin inhibitor cassette

SEQ ID NO: 31 Amino acid sequence of a SRC Homology 3 domain

SEQ ID NO: 32 Amino acid sequence of a protein binding partner-SRC Homology 3 domain-Binding protein cassette

SEQ ID NO: 33 Amino acid sequence of a SRC Homology 3 domain-binding protein cassette

SEQ ID NO: 34 Amino acid sequence of a binding protein-SRC Homology 3 domain protein binding partner cassette

SEQ ID NO: 35 Amino acid sequence of a binding protein-SRC Homology 3 domain cassette

SEQ ID NO: 36 Amino acid sequence of a binding protein-SRC Homology 3 domain mutant protein binding partner

SEQ ID NO: 37 Amino acid sequence of a modified binding protein-SRC Homology :

domain cassette

SEQ ID NO: 38 Amino acid sequence of a peptide mimetic p53 ^{17 28}

SEQ ID NO: 39 Amino acid sequence of a peptide mimetic PMI SEQ ID NO: 40 Amino acid sequence of a peptide mimetic PMI ^N8A

SEQ ID NO: 41 Amino acid sequence of a peptide mimetic PDI

SEQ ID NO: 42 Amino acid sequence of a modified binding protein-peptide mimetic p53 ^{17 28} cassette

SEQ ID NO: 43 Amino acid sequence of a modified binding protein-peptide mimetic

PMI cassette

SEQ ID NO: 44 Amino acid sequence of a modified binding protein-peptide mimetic

PMI ^N8A cassette

SEQ ID NO: 45 Amino acid sequence of a modified binding protein-peptide mimetic

PDI cassette

SEQ ID NO: 46 Amino acid sequence of a peptide mimotope of the CD20 epitope

SEQ ID NO: 47 Amino acid sequence of a mutant peptide mimotope of the CD20 epitope

SEQ ID NO: 48 Amino acid sequence of a binding protein-peptide mimotope of the

CD20 epitope -protein binding partner cassette

SEQ ID NO: 49 Amino acid sequence of a binding protein-mutant peptide mimotope ( the CD20 epitope -protein binding partner cassette

SEQ ID NO: 50 Amino acid sequence of a binding protein-peptide mimotope of the

CD20 epitope -mutant protein binding partner cassette

SEQ ID NO: 51 Amino acid sequence of a binding protein- mutant peptide mimotope the CD20 epitope - mutant protein binding partner cassette

SEQ ID NO: 52 Amino acid sequence of a modified binding protein-peptide mimotopi of the CD20 epitope cassette

SEQ ID NO: 53 Amino acid sequence of a modified binding protein-mutant peptide mimotope of the CD20 epitope cassette

SEQ ID NO: 54 Amino acid sequence of a peptide aptamer

SEQ ID NO: 55 Amino acid sequence of a cell penetrating peptide

SEQ ID NO: 56 Amino acid sequence of a modified binding protein-peptide aptamer cassette

SEQ ID NO: 57 Amino acid sequence of a binding protein - peptide aptamer - protein binding partner - cell penetrating peptide cassette

SEQ ID NO: 58 Amino acid sequence of a EGFR-Affybody

SEQ ID NO: 59 Amino acid sequence of a Folate Receptor alpha protein

SEQ ID NO: 60 Amino acid sequence of a Folate Receptor beta protein

SEQ ID NO: 61 Amino acid sequence of a Ephrin type A receptor 2

SEQ ID NO: 62 Amino acid sequence of a C-X-C chemokine receptor type 4 SEQ ID NO: 63 Amino acid sequence of a peptide linker

SEQ ID NO: 64 Amino acid sequence of a FITC-V5 -protein binding partner

SEQ ID NO: 65 Amino acid sequence of a Biotin-V5-protein binding partner

SEQ ID NO: 66 Amino acid sequence of a binding protein-peptide aptamer- protein binding partner cassette

SEQ ID NO: 67 Amino acid sequence of a binding protein - peptide aptamer - protein binding partner - cell penetrating peptide cassette

SEQ ID NO: 68 Amino acid sequence of a Epidermal growth factor receptor (EGFR)

SEQ ID NO: 69 Amino acid sequence of a Bim BH3 sequence

SEQ ID NO: 70 Amino acid sequence of a Bid BHS sequence

SEQ ID NO: 71 Amino acid sequence of a PUMA BH3 sequence

SEQ ID NO: 72 Amino acid sequence of a modified binding protein-BimBH3-cassette

SEQ ID NO: 73 Amino acid sequence of a modified binding protein-BidBH3-cassette

SEQ ID NO: 74 Amino acid sequence of a modified binding protein-PumaBH3 -cassette

SEQ ID NO: 75 Amino acid sequence of a binding protein-BimBH3-cassette

SEQ ID NO: 76 Amino acid sequence of a binding protein-BidBH3-cassette

SEQ ID NO: 77 Amino acid sequence of a binding protein-PumaBH3 -cassette

SEQ ID NO: 78 Amino acid sequence of a Histatin-1

SEQ ID NO: 79 Amino acid sequence of a modified binding protein-Hisl -cassette

SEQ ID NO: 80 Amino acid sequence of a binding protein-Hisl -binding partner protein cassette

SEQ ID NO: 81 Amino acid sequence of a binding protein-Hisl cassette

SEQ ID NO: 82 Amino acid sequence of a folate receptor beta protein

SEQ ID NO: 83 Amino acid sequence of a mutant FITC -protein binding partner

SEQ ID NO: 84 Amino acid sequence of a cell penetrating peptide -partner-peptide aptamer-binding protein cassette

SEQ ID NO: 85 Amino acid sequence of a cell penetrating peptide- mutant protein binding partner-peptide aptamer-binding protein cassette

SEQ ID NO: 86 Amino acid sequence of a partner -peptide aptamer-binding protein-cell penetrating peptide cassette

SEQ ID NO: 87 Amino acid sequence of a mutant protein binding partner-peptide

aptamer-binding protein-cell penetrating peptide cassette

SEQ ID NO: 88 Amino acid sequence of a protein binding partner-peptide aptamer- binding protein cassette

SEQ ID NO: 89 Amino acid sequence of a mutant protein binding partner-peptide

aptamer-binding protein cassette SEQ ID NO: 90 Nucleic acid sequence of BP library cassette

SEQ ID NO: 91 Nucleic acid sequence of MP library cassette

SEQ ID NO: 92 Amino acid sequence of a binding protein partner CPP

SEQ ID NO: 93 Amino acid sequence of a CPP binding protein partner

SEQ ID NO: 94 Nucleic acid sequence of RMP library cassette

SEQ ID NO: 95 Nucleic acid sequence of MPR library cassette

SEQ ID NO: 96 Amino acid sequence of a peptide linker

SEQ ID NO: 97 Amino acid sequence of a peptide mimetic p28

SEQ ID NO: 98 Amino acid sequence of albumin binding domain (ABD) variant

(ABDcon)

SEQ ID NO: 99 Amino acid sequence of ABD variant (ABDcon5)

SEQ ID NO: 100 Amino acid sequence of ABD variant (ABDcon9)

SEQ ID NO: 101 Amino acid sequence of ABD variant (SA20)

SEQ ID NO: 102 Amino acid sequence of Partner- ABDcon5

SEQ ID NO: 103 Amino acid sequence of Partner- ABDcon9

SEQ ID NO: 104 Amino acid sequence of Partner-SA20

SEQ ID NO: 105 Amino acid sequence of MABDcon

SEQ ID NO: 106 Amino acid sequence of MABDcon5

SEQ ID NO: 107 Amino acid sequence of MABDcon9

SEQ ID NO: 108 Amino acid sequence of MSA20.

SEQ ID NO: 109 Nucleic acid sequence of BP_BPP_libnuc

SEQ ID NO: 110 Amino acid sequence of BP_BPP_libprotein

SEQ ID NO: 111 Amino acid sequence of BP_BPP_libpeptide

SEQ ID NO: 112 NGS Amplicon PCR primer t-T7SpyCy_Fl

SEQ ID NO: 113 NGS Amplicon PCR primer t-T7SpyCy_F2

SEQ ID NO: 114 NGS Amplicon PCR primer t-T7SpyCy_F3

SEQ ID NO: 115 NGS Amplicon PCR primer t-T7SpyCy_F4

SEQ ID NO: 116 NGS Amplicon PCR primer t-T7SpyCy_Rl

SEQ ID NO: 117 NGS Amplicon PCR primer t-T7SpyCy_R2

SEQ ID NO: 118 NGS Amplicon PCR primer t-T7SpyCy_R3

SEQ ID NO: 119 NGS Amplicon PCR primer t-T7SpyCy_R4

SEQ ID NO: 120 M_Loop2_libnuc

SEQ ID NO: 121 M_Loop2_libprot

SEQ ID NO: 123 GSGAS-Linker

SEQ ID NO: 124 QGTGS-Linker

SEQ ID NO: 125 ABDcore sequence SEQ ID NO: 126 Amino acid sequence of modified binding protein p28 cassette (Mp28)

SEQ ID NO: 127 Amino acid sequence of binding protein p53 ^{17 28} cassette (Bp53 ^{17 2S} )

SEQ ID NO: 128 Amino acid sequence of binding protein PMI ^N8A cassette (BPMI ^N8A )

SEQ ID NO: 129 Amino acid sequence of binding protein p28 cassette (Bp28)

SEQ ID NO: 130 Amino acid sequence of ABD N-terminal extension

Brief Description of Drawings

Figure 1 is a graphical representation of showing the inhibition of the proteolytic activity of trypsin upon addition of PBB or BBP.

Figure 2 is a graphical representation of showing the inhibition of the proteolytic activity of trypsin upon addition of MB.

Figure 3 is a schematic overview of the modified binding protein and binding protein p53-MDM2/ MDMX/ COPl peptide inhibitor fusion protein constructs and the various p53-MDM2/ MDMX/ COPl peptide inhibitor sequences.

Figure 4 shows an image of an SDS-PAGE gel analysis of modified binding protein-p53-MDM2/ MDMX/ COPl peptide inhibitor fusion protein ligation. The various fusion protein constructs retain the ability to ligate to a protein binding partner in trans.

Figure 5 shows an image of an SDS-PAGE gel analysis of binding protein p53- MDM2/ MDMX/ COPl peptide inhibitor fusion protein ligation. The fusion protein retains the ability to ligate to a fused protein binding partner in cis.

Figure 6 is a graphical representation of showing the 48-hour Presto Blue

17 28 measured cell viabilities of T47D and CHO-Kl cells upon the addition of Mp53

17 28

and Mp53 ^" -CPP-partner conjugate.

Figure 7 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D cells upon the addition of MPMI ^N8A , MPMI ^N8A -CPP- partner conjugate, MPDI and MPDI-CPP-conjugate.

Figure 8 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of Mp28 and Mp28-CPP-conjugate.

Figure 9 is a graphical representation of showing the 48-hour Presto Blue

17 28 measured cell viabilities of T47D and CHO-Kl cells upon the addition of Bp53 ^" and

17 28

Bp53 ^" -CPP-partner conjugate.

Figure 10 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D cells upon the addition of BPMI ^N8A and BPMI ^N8A - CPP-partner conjugate. Figure 11 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of Bp28 and Bp28-CPP-partner conjugate.

Figure 12 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of CPP-partner (negative control) and PBS Buffer.

Figure 13 shows results of an assay to determine the ability of MABDcon, MABDcon5, MABDcon9, and MSA20 to retain the specific ABD interaction with the protein binding partner, by test with FITC-partner. MABDcon, MABDcon5, MABDcon9, and MSA20 retain the ability to ligate a FITC-partner.

Figure 14 shows results of an interferometry assay to determine the affinity of biotinylated human serum albumin (HSA) for each of MABDcon, MABDcon5, MABDcon9, and MSA20.

Figure 15 shows results of an interferometry assay to determine the affinity of biotinylated human serum albumin (HSA) for each of BP+PBP+ABDcon5; BP+PBP+ABDcon9; and BP alone (negative control).

Figure 16 is a schematic overview of possible configurations of Phylomers in a phylomer phage display library constrained by an interaction between binding protein and protein binding partner. Phylomer cyclisation is achieved through conjugation of a fused protein binding partner in cis.

Figure 17 is a schematic overview of possible configurations of Phylomers in a phylomer phage display library constrained by modified binding protein loop display. Phylomers are inserted for cyclisation within Loop2 of the modified binding protein, leaving the modified binding protein available for conjugation with a protein binding partner in trans.

Fig. 18 is a schematic overview of a cyclised phylomer library enrichment strategy. Addition of magnetic bead-bound protein binding partner results in conjugation to phage displaying properly folded modified binding-protein-phylomer fusion proteins, which can then be magnetically separated from phage displaying partial or improperly folded modified binding protein.

Fig. 19 is a bar graph summarising the results of the enrichment scheme depicted in Fig. 18. Following reaction of the modified binding protein Phylomer library with protein binding partner magnetic beads, the library is partially enriched for phage having read through in frame modified binding protein-Phylomer fusion proteins. Detailed Description

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth.

Each example of the present disclosure described herein is to be applied mutatis mutandis to each and every other example unless specifically stated otherwise.

Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such techniques are described and explained throughout the literature in sources such as Perbal 1984, Sambrook et ah, 2001, Brown (editor) 1991, Glover and Hames (editors) 1995 and 1996, Ausubel et al. including all updates until present, Coligan et al. (editors) (including all updates until present), Maniatis et al. 1982, Gait (editor) 1984, Hames and Higgins (editors) 1984, Freshney (editor) 1986.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning. The term "about", unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, of the designated value. For the avoidance of doubt, the term "about" followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, "about 10" also encompasses 10 exactly).

The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. hopeptide bond

The binding protein and protein binding partner disclosed herein are capable of forming an isopeptide bond with each other. The term "isopeptide bond" refers to a covalent amide bond formed between a first reactive group and a second reactive group. The first reactive group is a carboxyl group and the second reactive group is an amino group. In one example, the first reactive group is a lysine residue and the second reactive amino acid is selected from the group consisting of an asparagine, an aspartic acid, a glutamine, or a glutamic acid residue. Accordingly, in one example, an isopeptide bond is formed between a lysine residue and an asparagine residue. In another example, an isopeptide bond is formed between a lysine residue and an aspartic acid residue. In another example, an isopeptide bond is formed between a lysine residue and a glutamine residue. In yet another example, an isopeptide bond is formed between a lysine residue and a glutamic acid residue. Two proteins linked via an intermolecular isopeptide bond are defined as complex.

Isopeptide bond formation may be an enzyme-dependent processes. Thus, the formation of an isopeptide bond may require an enzyme. Any enzyme known in the art to that catalyzes the formation of an isopeptide bond may be used. For example, the enzyme may be a transglutaminase.

Isopeptide bond formation may be an autocatalytic reaction. The isopeptide bond may therefore form autocatalytically e.g. without the presence of an enzyme catalyst or other agent. The presence of an isopeptide bond may be detected by suitable methods known in the art. For example, mass spectrometry is routinely used to detect an isopeptide bond since the formation of the isopeptide bond results in a loss of a water molecule. In the context of an intermolecular isopeptide bond, an 18 kDa shift in the molecular mass of a complex may be detected as compared to the combined molecular mass of the binding protein and protein binding partner as determined separately.

Binding protein

The binding protein is capable of forming an intermolecular isopeptide bond with any protein binding partner described herein. For example, the binding protein comprises the first reactive group and the protein binding partner comprises the second reactive group. In one example, the binding protein comprises a lysine residue {i.e. the first reactive group) and the protein binding partner comprises an amino acid residue selected from the group consisting of an asparagine, an aspartic acid, a glutamine, or a glutamic acid residue {i.e. the second reactive group). Accordingly, in one example, an isopeptide bond is formed between a lysine residue of the binding protein and an asparagine residue of the protein binding partner. In another example, an isopeptide bond is formed between a lysine residue of the binding protein and an aspartic acid residue of the protein binding partner. In another example, an isopeptide bond is formed between a lysine residue of the binding protein and a glutamine residue of the protein binding partner. In yet another example, an isopeptide bond is formed between a lysine residue of the binding protein and a glutamic acid residue of the protein binding partner. Alternatively, the protein binding partner comprises the first reactive group and the binding protein comprises the second reactive group.

Isopeptide bond formation may occur almost immediately after contact between the binding protein and the protein binding partner. For example, an isopeptide bond may form within at least 1 minute or at least 2 minutes or at least 3 minutes or at least 4 minutes or at least 5 minutes or at least 10 minutes or at least 15 minutes or at least 20 minutes or at least 25 minutes or at least 30 minutes of contacting the binding protein with the protein binding partner. In another example, an isopeptide bond may form within at least 1 hour or at least 2 hours or at least 4 hours or at least 6 hours or at least 8 hours or at least 12 hours or at least 16 hours or at least 20 hours or at least 24 hours of contacting a binding protein with a protein binding partner.

Isopeptide bond formation between a binding protein and a protein binding partner may occur under any conditions. For example, an isopeptide bond may form in phosphate-buffered saline (PBS) at pH 7.0 and at 25°C. In another example, an isopeptide bond may form in phosphate-buffered saline (PBS) at pH 7.0 and at 4°C.

A complex comprising a binding protein and a protein binding partner linked via an isopeptide bond may stable under conditions that may dissociate non-covalently linked proteins. Thus, a complex may be stable at a high temperature. For example, a complex may be stable at 80°C or at 85°C or at 90°C or at 95°C or at 100°C. Alternatively, or in addition, a complex may be stable when subjected to chemical treatment. For example, complex may be stable at pH 2 or at pH 3.

A binding protein may be of any length. For example, a binding protein may comprise or consist of at least 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300 or 350 or more amino acids. A binding protein may therefore comprise or consist of from 80 to 150 amino acids, such as from 90 to 140 amino acids, or such as from 100 to 130 amino acids, including any length within said range(s).

A binding protein may have an amino acid sequence that is substantially similar to the amino acid sequence set forth in SEQ ID NO: 1. By "substantially similar" it is meant that the binding protein may comprise or consist of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. For example, a binding protein may comprise or consist of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In another example, a binding protein may comprise or consist of an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In another example, a binding protein may comprise or consist of an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In another example, a binding protein may comprise or consist of an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In this example, the binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO 2.

Alternatively, a binding protein may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 14. For example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 4.

In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 5. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 6. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 7. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 8. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 9. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 10. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 11. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 12. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 13. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 14.

Percentage amino acid sequence identity with respect to a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Amino acid sequence identity may be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk/Tools/emboss/align/. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix "Blosum62" is utilized for amino acid sequences and the default matrix.

A binding protein may be a fragment of an isopeptide protein e.g. a protein which comprises an intramolecular isopeptide bond. Several isopeptide proteins comprising an intramolecular isopeptide bond are known in the art and are described, for example in Kang et al. 2007.

In one example, a binding protein is linked to a target.

In one example, a binding protein is linked to a molecule.

In another example, a binding protein is linked to a cell penetrating peptide.

Protein binding partner

The protein binding partner is capable of forming an intermolecular isopeptide bond with any binding protein described herein. In one example, the protein binding partner comprises the first reactive group and the binding protein comprises the second reactive group of an intermolecular isopeptide bond. In one example, the binding protein comprises the first reactive group and the protein binding partner comprises the second reactive group of an intermolecular isopeptide bond.

The protein binding partner may be a fragment of an isopeptide protein. The protein binding partner may be of any length. For example, the protein binding partner may comprise or consist of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more amino acids. The protein binding partner may therefore comprise or consist of from 8 to 30 amino acids, such as from 8 to 20 amino acids, or such as from 8 to 15 amino acids, including any length within said range(s). In another example, the protein binding partner may consist of or may comprise from 5 to 10 amino acids.

The protein binding partner may comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. For example, the first sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one example, a protein binding partner may comprise or consist of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In another example, a protein binding partner may comprise or consist of an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

Alternatively, the protein binding partner may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 16-18. For example, the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 16. In another example, the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 17. In another example, the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 18.

In one example, a protein binding partner is linked to a target

In one example, a protein binding partner is linked to a molecule.

In another example, a protein binding partner is linked to a cell penetrating peptide.

Peptide

The term "peptide" is intended to include compounds composed of amino acid residues linked by amide bonds. A peptide may be natural or unnatural, ribosome encoded or synthetically derived. Typically, a peptide will consist of between 2 and 200 amino acids. For example, the peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within said range(s). The peptide may comprise or consist of fewer than about 150 amino acids or fewer than about 125 amino acids or fewer than about 100 amino acids or fewer than about 90 amino acids or fewer than about 80 amino acids or fewer than about 70 amino acids or fewer than about 60 amino acids or fewer than about 50 amino acids.

Peptides include peptide analogues, peptide derivatives and peptidomimetic. Examples of peptide analogues include peptides comprising one or more non-natural amino acids. Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatized. In one example, a peptidomimetic s include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules, "inverso" peptides in which all L-amino acids are substituted with the corresponding D- amino acids, "retro-inverso" peptides in which the sequence of amino acids is reversed and all L-amino acidsare replaced with D-amino acids and other isosteres, such as peptide back-bone (i.e., amide bond) mimetics, including modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including \|/[CH ₂ S]\|/, \|/[CH ₂ H], \|/[CSNH ₂ ], \|/[NHCO], \|/[COCH ₂ ], and ψ[(Ε) or (Z) CH=CH]. In the nomenclature used above, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets. Other possible modifications include an N-alkyl (or aryl) substitution ^[CO R]), backbone crosslinking to construct lactams and other cyclic structures, and other derivatives including C-terminal hydroxymethyl derivatives, O-modified derivatives and N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.

Peptides may be encoded by nucleic acid fragments of genomic DNA or cDNA obtained from an evolutionary diverse range of organisms from Viruses, Bacteria, Archaea, and Eukarya. For example, nucleic acid fragments may be obtained from Aeropyrum pernix, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Synechocystis PCC 6803, Thermoplasma volcanium and Thermotoga maritima. Nucleic acid fragments may be generated using one or more of a variety of methods known to those skilled in the art. Suitable methods include, as well as those described in the examples below, for example, mechanical shearing (e.g. by sonication or passing the nucleic acid through a fine gauge needle), digestion with a nuclease (e.g. Dnase 1), partial or complete digestion with one or more restriction enzymes, preferably frequent cutting enzymes that recognize 4-base restriction enzyme sites and treating the DNA samples with radiation (e.g. gamma radiation or ultra-violet radiation).

A peptide may be linked to one or more peptide linkers to facilitate insertion of the peptide into a region of a binding protein. For example, a peptide may linked at a first end to a peptide linker and at a second end to a peptide linker.

A peptide may be a cell penetrating peptide.

Complex

In one example, a complex comprises any binding protein described herein, any peptide described herein and any protein binding partner described herein, wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond, the binding protein is linked a terminus of the peptide and the peptide tag is linked to another terminus of the peptide. In one example, the binding protein is linked the N' terminus of the peptide and the peptide tag is linked to the C terminus of the peptide. In one example, the binding protein is linked to the C terminus of the peptide and the peptide tag is linked to the N' terminus of the peptide.

In one example, a complex comprises any modified binding protein described herein and any protein binding partner described herein, wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond.

In one example, any complex described herein is displayed on a bacteriophage. In one example, the bacteriophage is a T phage. In another example, the bacteriophage is a filamentous phage. In another example, the bacteriophage is a lysogenic bacteriophage. In another example, the bacteriophage is a lambda phage.

In another example, any complex described herein is displayed on a nanoparticle or a microparticle.

In another example, any complex described herein is displayed within in a cell. In one example, the cell is a bacterial cell. In another example, the cell is a yeast cell. In another example, the cell is a mammalian cell. Modified binding protein

Any binding protein described herein may be modified. The term "modified binding protein" refers to a binding protein wherein one or more peptides are inserted into one or more regions of the binding protein. The term "region" refers to one or more subsequences within a protein. Typically, a protein contains multiple distinct regions. Each region may contain sites for inserting a peptide with respect to a reference sequence. For example, the region may comprise or consist of amino acid residues 12-22 of SEQ ID NO: 1. For example, the region may comprise or consist of amino acid residues 12-22 of SEQ ID NO: 1. Thus, the region may comprise or consist of amino acid residues 12-16 or 17- 21 or 13-20, or 14-19 or 15-18 or 12-13 or 13-14 or 14-15 or 15-16 or 16-17 or 17-18 or 18-19 or 19-20 or 20-21. In another example, the region may comprise or consist of amino acid residues 27-31 of SEQ ID NO: 1. A region may therefore comprise or consist of amino acid residues 27-28 or 28-29 or 29- 30 or 30-31. In another example, the region may comprise or consist of amino acid residues 38-43 of SEQ ID NO: 1. A region may therefore comprise or consist of amino acid residues 39-41 or 38-39 or 39-40 or 40-41 or 41-42 or 42-43. In another example, the region may comprise or consist of amino acid residues 48-50 of SEQ ID NO: 1. A region may therefore comprise or consist of amino acid residues 48-49 or 49-50. In another example, the region may comprise or consist of amino acid residues 59-68 of SEQ ID NO: 1. A region may therefore comprise or consist of amino acid residues 60- 67 or 61-66 or 62-65 or 59-60 or 60-61 or 61-62 or 62-63 or 63-64 or 64-65 or 65-66 or 66-67 or 67-68.

A modified binding protein may be displayed on a particle. Various in vitro methods for displaying proteins on particles are are known in the art and described, for example, in Ullman et al. 2011. For example, the in vitro display method used may be a ribosome display, a covalent display or a mRNA display.

In one example, a modified binding protein is displayed on a ribosome.

In one example, a modified binding protein is displayed on a RepA protein

In one example, a modified binding protein is displayed on a DNA puromycin linker.

In one example, a modified binding protein is displayed on a RNA puromycin linker.

In one example, the particle may be a bacteriophage. The bacteriophage may be labelled with fluorescent tag or a fluorophore. The bacteriophage may be a filamentous phage. For example, the filamentous phage may be a M13 phage or a fl phage or a fd phage or a IKe phage or a Ifl or a If2 phage. The filamentous phage may be M13. In another example, the bacteriophage may be a lysogenic bacteriophage. In another example, the bacteriophage may be a lambda phage. In another example, the bacteriophage may be a T phage. For example, the T phage may a T3 phage or a T4 phage or a T7 phage. The T phage may be a T7 phage. Typically, a protein to be displayed on a bacteriophage is linked to a coat protein of the bacteriophage phage. For filamentous phage, the coat protein may be a pill coat protein or a pVI coat protein or a pVII coat protein or a pVIII coat protein or a pIX coat protein.

In another example, the particle may be a magnetic particle, a nanoparticle or a microparticle. Various methods of displaying a protein on a magnetic particle, a nanoparticle or a microparticle have been used including, for example, electrostatic assembly as described, for example, in Goldman et al. 2002, covalent cross-linking as described, for example, in Gao et al. 2004, avidin-biotin technology as described, for example, in Gref et al. 2003, or membrane integration as described, for example, in Mirzabekov et al. 2000. The person skilled in the art will understand that the amount and stability of a displayed protein is dependent on the method used for coupling.

A modified binding protein may be displayed within a cell. In one example, a modified binding protein may be displayed within a bacterial cell. In another example, modified binding protein may be displayed within a yeast. In one example, modified binding protein may be displayed within a mammalian cell.

A modified binding protein may be displayed in a cell free system. In one example, a modified binding protein may be displayed in an in vitro display system such as ribosome display, mRNA display, covalent display or CIS display.

Peptide constraint

In one example, a peptide inserted into a region of a modified binding protein may be constrained. Similarly, a peptide linked at a first end to a binding protein and at a second end to a protein binding partner may be constrained. By "constrained" it is meant that the peptide when inserted into a region of a modified binding protein or when a binding protein is linked to the protein binding partner via a covalent isopeptide bond wherein a peptide when linked at a first end to a binding protein and at a second end to a protein binding partner, has a reduced degree of freedom as compared to the corresponding linear peptide.

The degree of freedom may be a rotational degree of freedom. The degree of freedom may be a translation degree of freedom. The degree of freedom may be a vibrational degree of freedom. The degree of freedom of a peptide can be measured with any method known in the art as described, for example in Minary and Levitt, 2010.

In one example, circular dichroism (CD) spectroscopy is used to measure the degree of freedom of a peptide. CD is a fast and relatively easy spectroscopic technique to study protein conformational behaviour as described, for example, in Siligardi et al. 2014. Through pattern recognition of the CD spectral features, proteins can be classified in terms of their folding and secondary structure composition. CD may be used for proteins have proven difficult to crystallise for use in X-ray crystallographic structural determination or too complex for NMR structural studies.

In another example, nuclear magnetic resonance (NMR) spectroscopy is used to measure the degree of freedom of a peptide. NMR spectroscopy produces information on the relative positions of all atoms including hydrogen for a given protein. As such, NMR spectroscopy is an excellent tool to determine macromolecular structure at atomic resolution. In addition, NMR spectroscopy can follow protein folding as it happens, either by fast data acquisition of heteronuclear single quantum coherence or correlation experiments or by H/D exchange as described, for example, in Bieri et al., 2011.

In another example, the degree of freedom of a peptide is determined by crystallography. X-ray crystallography produces information on the relative positions of all non-hydrogen atoms for a given protein as described, for example, in Wlodawer et al. 2013.

In one example, a constrained peptide is capable of forming a stable secondary structure and/or conformation sufficient for binding and/or internalization and/or localization to a subcellular compartment e.g., without a need for intramolecular disulphide bridge formation to produce a loop.

Molecule

The term "molecule" refers to any compound. For example, a molecule may be a ligand, a carbohydrate, a polymer, an enzyme, a cellular receptor, an antigen mimic of a cellular receptor, a cell or a cell surface protein. In one example, a molecule is a detectable label. In another example, a molecule is a therapeutic agent. In another example, the molecule is a toxin. In another example, the molecule is a cell surface receptor. In another example, the molecule is an intracellular receptor. In another example, the molecule is a transcription factor. In another example, the molecule is an enzyme. In another example, the molecule is a ligand. For example, the ligand may be a surface receptor ligand, an intracellular receptor ligand or a target ligand. In another example, the molecule is an antigen. In another example, the molecule is a nucleic acid therapeutic. In another example, the molecule is short activating RNA.

Any molecule described herein that has a molecule weight of less than 1000 Da is described herein as a "small molecule".

Detectable label

The term "detectable label" refers to any type of molecule which can be detected by optical, fluorescent, isotopic imaging or by mass spectroscopic techniques, or by performing simple enzymatic assays. Any detectable label known in the art may be used. The detectable label may be toxic to cells or cytotoxic. Accordingly, the detectable label may also be a therapeutic agent or a cytotoxic agent. The detectable label may be selected form the group consisting of a fluorescent tag, a chemical tag, an epitope tag, a isobaric tagor a radiochemical tag.

A fluorescent tag may be a fluorophore. For example, a fluorophore may be fluorescein isothiocyante, fluorescein thiosemicarbazide, rhodamine, Texas Red, a CyDye such as Cy3, Cy5 and Cy5.5, a Alexa Fluor such as Alexa488, Alexa555, Alexa594 and Alexa647) or a near infrared fluorescent dye. A fluorescent tag may be a fluorescent protein. For example, a fluorescent protein may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), AcGFP or TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTFPl (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellowl, mBanana, Kusabira,ange, mOrange, dTomato, dTomato-Tandem, AsRed2, mRFPl, Jred, mCherry, HcRedl, mRaspberry, HcRedl, HcRed-Tandem, mPlum, AQ 143. A fluorescent tag may be a quantum dot. Fluorescent tags may be detected using fluorescent microscopes such as epifluorescence or confocal microscopes, fluorescence scanners such as microarray readers, spectrofluorometers, microplate readers and/or flow cytometers.

A chemical tag may be SNAP tag, a CLIP tag, a HaloTag or a TMP-tag. In one example, the chemical tag is a SNAP-tag or a CLIP-tag. SNAP and CLIP fusion proteins enable the specific, covalent attachment of virtually any molecule to a protein of interest as described, for example, in Correa 2015. In another example, the chemical tag is a HaloTag. HaloTag involves a modular protein tagging system that allows different molecules to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells as described, for example, in Los et al. 2008. In another example, the chemical tag is a TMP-tag. TMP-tags are able to label intracellular, as opposed to cell-surface, proteins with high selectivity as described, for example, in Chen et al. 2012.

An epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag. Epitope tags are routinely detected with commercially available antibodies. A person skilled in the art will be aware that an epitope tag may facilitate purification and/or detection. For example, a conjugate containing a hexahistidine tag may be purified using methods known in the art, such as, by contacting a sample comprising the protein with nickel- nitrilotriacetic acid (Ni-NTA) that specifically binds a hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to an epitope tag may be used in an affinity purification method.

An isobaric tag may be a mass tag or an isobaric tag for relative absolute quantification (iTRAQ). A mass tag is a chemical label used for mass spectrometry based quantification of proteins and peptides. In such methods mass spectrometers recognise the mass difference between the labeled and unlabeled forms of a protein or peptide, and quantification is achieved by comparing their respective signal intensities as described, for example, in Bantscheff et al. 2007. Examples of mass tags include TMTzero, TMTduplex, TMTsixplex and TMT 10-plex. An isobacric tag for relative absolute quantification (iTRAQ) is a chemical tag used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment as described, for example, in Wiese et al. 2007.

A radiochemical tag may be a γ-emitting radionuclide, Auger-emitting radionuclide, β-emitting radionuclide, an a-emitting radionuclide, or a positron- emitting radionuclide. The radionuclides may be 3H, 14C, 32P, 33P, 35S, 1251 or 51Cr. The detectable labels may be selected from the group consisting of biotin, a acyl carrier protein tag, or streptavidin. Radiochemical tags may be detected with an ionization chamber or an autoradiograph.

Other types of detectable labels include biotin, an acyl carrier protein tag, or streptavidin.

Therapeutic agent

The term "therapeutic agent" refers to any type of molecule capable of having a biological effect. A therapeutic agent may be selected form the group consisting of a therapeutic compound, a chemotherapeutic agent or a cytotoxic agent. A therapeutic compound may induce an immune response. For example, the therapeutic compound may be, a chemokine (e.g., BCA-1, BRAK, CTACK, CXCL17, ENA 78, Eotaxin, Interleukine-8, MCP, Platelet Factor-4 and Rantes), a cytokine (e.g., tumor necrosis factor alpha, Interferon, Beta Defensin, Betacellulin, Leukemia Inhibitory Factor, Hedgehog Protein, Follistatin, Flt3 Ligand, Granulocyte-Macrophage Colony-Stimulating Factor), a growth factor (e.g., Growth Hormone, Colony Stimulating Factor, Epidermal Growth Factor, Erythropoietin, Myostatin, RANK Ligand, Osteoprotegerin, Noggin, and VEGF ), a hormone (e.g., Endothelin, Exendin, FSH, Stanniocalcin, Thymosin), a human leukocyte antigen, a co- stimulatory molecule or a tumor-associated antigen. In one example, the therapeutic compound is an enzyme.

A chemotherapeutic agent may be an alkylating agent, a kinase inhibitor, a vinca alkaloid, anthracycline, an anti-metabolite, an aromatase inhibitor or a topoisomerase inhibitor.

A cytotoxic agent may affect cell division. In one example, the cytotoxic agent may affect the S phase of the cell cycle. Examples of cytotoxic agents that affect the S phase of the cell cycle include Ergot alkaloids and Methotrexate. In another example, the cytotoxic agent may affect the G2 phase of the cell cycle. Examples of cytotoxic agents that affect the G2 phase of the cell cycle include Etoposide and Bleomycin. In another example, the cytotoxic agent may affect the M phase of the cell cycle. Examples of cytotoxic agents that affect the M phase of the cell cycle include Gresiofulvin, Paclitaxel, Vincristine and Vinblastine. Alternatively, or in addition, a cytotoxic agent may induce cell death. Any art recognized method may be employed to determine the viability of the cells. For example, determining viability of the cell may comprise determining the doubling rate of the cell e.g., the period of time required for the cell to divide e.g., nucleic acid content or cell counting such as by FACS.

Toxin

The term "toxin" refers to any molecule capable of causing cell death or impaired cell survival when internalised in a cell. For example, a toxin may induce cell death in more than 50% or more 60% or more than 70% or more than 80% or more than 90% or more than 95% or more than 97% or more than 98% or more than 99% of cells in which it is internalized.

In one example, the toxin comprises one or more domains from plant, bacterial or fungal protein toxins. In another example, the toxin may classified according to their mechanism of action and/or structural organization, such as, for example, ADP- ribosylating toxins; N-glycosidase containing ribosome inactivating toxins; and binary bacterial toxins that comprise separate cell binding and catalytic domains, including, for example, anthrax toxin, pertussis toxins, cholera toxin, E. coli heat-labile enterotoxin, Shiga toxin, pertussis toxin, Clostridium perfringens iota toxin, Clostridium spiroforme toxin, Clostridium difficile toxin, Clostridium botulinum C2 toxin, and Bacillus cereus vegetative insecticidal protein.

Methods to determine cell viability or cytotoxicity are known in the art such as, for example, plate viability assays, colony regression assays, plating assays, and fluorometric/colorimetric growth indicator assays based on detection of metabolic activity. In one example, cell viability is determined based on the ability of the membrane of viable test cells to exclude dyes, such as, for example, trypan blue or propidium iodide. Living test cells exclude such dyes and do not become stained. In contrast, dead or dying test cells that have lost membrane integrity allow these dyes to enter the cytoplasm and stain various compounds or organelles within the test cell. A number of cell viability assays and cytotoxicity assays are also commercially available. Diagnostic/Theranostic

The term "diagnostic" refers to one or more components, complexes, and/or biomarkers the detection or level of which is indicative of a specific health condition {e.g., the presence or absence of a cancer). The term "theranostic" refers to one or more agents that combine a therapeutic modality with a diagnostic modality to facilitate the assessment of disease status following administration of the theranostic agent.

Target

The term "target" refers to any substance expressed by a cell, on a cell or present within a cell, to which a peptide may bind. For example, a target may be a ligand, a carbohydrate, a polymer, an enzyme, a cellular receptor or a cell surface protein. A target may be expressed by specific cells. For example, the target may be expressed by tumor cells, immune cells, bacterial cells, fungal cells, parasites, or virus infected cells.

In one example, the target is expressed by tumor cells. In another example, the target is expressed by bacterial cells. In another example, the target is expressed by fungal cells. In another example, the target is expressed by parasites. In another example, the target is expressed by virus infected cells.

A target may be expressed in a specific region of a cell. In one example, the target is expressed in a subcellular compartment. In another example, the target is a cell surface receptor. In another example, the target is an intracellular receptor.

A target may be a transcription factor. A target may be a signalling adaptor protein or a component thereof. Examples of signalling adaptor proteins include, for example, GRAP, GRAP2, LDLRAP1, NCK1, NCK2, NOS 1AP, PIK3AP1, SH2B 1, SH2B2, SH2B3, SH2D3A, SH2D3C, SHB, SLC4A1AP, GAB 2.

Subcellular compartment

The term "subcellular compartment" refers to any compartment in a cell including, but not limited to cytosol, endosome, nucleus, endoplasmic reticulum, golgi, vacuole, mitochondrion, plastid such as chloroplast or amyloplast or chromoplast or nucleus, cytoskeleton, centriole, microtubule-organizing center (MTOC), acrosome, glyoxysome, melanosome, myofibril, nucleolus, peroxisome, nucleosome or microtubule or the cytoplasmic surface such the cytoplasmic membrane or the nuclear membrane. Cell surface receptor

The term "cell surface receptor" refers to any protein that is present on the surface of a cell and is capable of transmitting or transducing a signal in the cell. Any cell surface receptor known in the art may be used such as those described, for example, in Uings and Farrow, 2000. For example, the cell surface receptor may be a ligand-gated ion channel receptor, an enzyme-coupled receptor or a G-protein-coupled receptor.

In one example, the cell surface receptor is a capable of binding and internalising any molecule via receptor mediated endocytosis.

In another example, the cell surface receptor the epithelial growth factor receptor (EGFR). For example, an epithelial growth factor receptor may be linked to an affibody and comprise the amino acid sequence set forth in SEQ ID NO: 58. In another example, the cell surface receptor is an Ephrin receptor. For example, the Ephrin receptor is Ephrin receptor EphA2 In another example, the cell surface receptor is C-X- C chemokine receptor type 4 (CXCR4). In another example, the cell surface receptor is folate receptor alpha protein. For example, the folate receptor alpha protein may comprise the amino acid sequence set forth in SEQ ID NO: 59. In another example, the cell surface receptor is a folate receptor beta protein. For example, the folate receptor beta protein may comprise the amino acid sequence set forth in SEQ ID NO: 82.

In another example, a synthetic cell surface receptor may be used. Examples of synthetic cell surface receptors are described for example in Hymel and Peterson 2010. Intracellular receptor

The term "intracellular receptor" refers to any protein located in a subcellular compartment that is capable of being activated by a ligand. In one example, the intracellular receptor is a nuclear receptor. In one example, the intracellular receptor is a constitutive androstane receptor. In one example, the intracellular receptor is a famesoid X receptor. In one example, the intracellular receptor is a IP3 receptor. In one example, the intracellular receptor is a liver X receptor. In one example, the intracellular receptor is a peroxisome proliferator-activated receptors. In one example, the intracellular receptor is a pregnane X receptor. In one example, the intracellular receptor is a retinoic acid receptor. In one example, the intracellular receptor is a retinoid X receptor.

Transcription factor

The term "transcription factor" refers to any protein involved in the process of converting, or transcribing, DNA into RNA. A transcription factor may bind DNA alone or a transcription factor may form complexes with other transcription factors and thus may bind DNA directly or indirectly.

In one example, the transcription factor is an activator. In one example, the transcription factor is a transcriptional co-activator. In one example, the transcriptional co-activator may be beta catenin. In one example, the transcriptional co-activator may be Master Mind Like Protein (MAML). In another example, the transcription factor is a repressor. In another example, the transcription factor is a co-repressor. In another example, the transcription factor is an initiator of transcription.

In one example, the transcription factor is STAT (signal transducers and activators of transcription) family protein. STAT family proteins are well known in the art and are described, for example, in Akira 1999. In one example, the STAT family protein is selected from the group consisting of STAT3 and STAT5.

In another example, the transcription factor is c-Myc. c-Myc is known to play a pivotal role in growth control, differentiation and apoptosis, and its abnormal expression is associated with many tumors as described, for example, in Hoffman and Liebermann 2008.

RNA binding protein

The term "RNA binding protein" refers to any protein involved in binding specifically to RNA. An RNA binding protein may bind to RNA alone or a may form complexes with other proteins or nucleic acids and thus may bind RNA directly or indirectly. In one example, the RNA binding protein is YB-1. YB-1 is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation as described, for example, in Bobkova et al. 2005.

Conjugate

The term "conjugate" refers a molecule comprising two or more elements or components that are linked by whatever means including chemical conjugation or recombinant means. In one example, a conjugate may comprise a binding protein linked to a molecule. In another example, a peptide linked to a molecule. Methods of conjugation are known in the art. Two elements or components may be linked together directly. The person skilled in the art will understand that the co-linear, covalent linkage of two or more proteins via their individual peptide backbones and expressed as a single molecule encoding those proteins forms a specific type of conjugate described herein as a "fusion protein". Two elements or components may be linked to each other via one or more peptide linkers.

Cell penetrating peptide

The term "cell penetrating peptide" refers to a peptide that is capable of crossing a cellular membrane. In one example, a cell penetrating peptide is capable of translocating across a mammalian cell membrane and entering into a cell. In another example, a cell penetrating peptide may direct a conjugate to a desired subcellular compartment. Thus, a cell penetrating peptide may direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal or nuclear membrane. A cell penetrating peptide may direct a molecule of interest from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively or in addition, a cell penetrating peptide may direct a molecule of interest across the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal and/or pulmonary barriers.

RNAi agent

The term "RNAi agent" refers to an RNA molecule having a structure characteristic of molecules that can inhibit transcription and/or translation a gene. Various RNAi agents are known in the art and are described, for example, in Kim and Rossi, 2009. Suitable RNAi agent include, for example, small interfering RNAs (siRNA), double stranded RNAs (dsRNAs), inverted repeats, short hairpin RNAs (shRNAs), small temporally regulated RNAs (stRNAs), clustered inhibitory RNAs (cRNAs), including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, and complex or compound clustered inhibitory RNA, dicer substrates, DNA-directed RNAi (ddRNAi), microRNA (miRNA), miRNA antagonists, microRNA mimics, microRNA agonists, blockmirs, microRNA mimetics, microRNA addbacks, and supermiRs.

Peptide linker

A peptide linker refers to amino acid sequences that connect, join or link two protein sequences. A peptide linker may be of any length. For example, the peptide linker may comprise or consist of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. The peptide linker may therefore comprise or consist of from 2 to 10 amino acids, such as from 2 to 8 amino acids, or such as from 4 to 6 amino acids. Thus, the peptide linker may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 20 to 23 or 63. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 20. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 21. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 23. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 63.

Vector

The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. For example, the vector may a bacterium, a plasmid, a bacteriophage, a cosmid, an episome or a virus. When the vector directs the expression of a gene encoding a protein or RNA operably connected thereto, the vector is called an "expression vector." Selection of appropriate vectors is within the knowledge of those having skill in the art. Signal peptide

The term "signal peptide" refers to a peptide sequence that directs the transport of a protein. Any signal peptide known in the art may be used. For example, a signal peptide may direct a protein through a secretory pathway of a cell. Alternatively, a signal peptide may bind to an extracellular domain of a receptor on an exterior surface of a cell. Upon internalization of the receptor, the signal peptide may facilitate the localization of a protein to a subcellular compartment. In another example, a signal peptide may be capable of directing a protein to a desired subcellular compartment of a cell. For example, a peptide signal may be a nuclear localisation signal. Several nuclear localisation signals are known in the art and are described for example by Emmott et al. 2009. Alternatively, a peptide signal may be a golgi localisation sequence. In another example, peptide signal may be a mitochondria localisation sequence.

A signal peptide may be a prokaryotic signal peptide. For example, the signal peptide may be a DsbA signal peptide, a TorT signal peptide, a TolB signal peptide or a Sfm signal peptide, a Lam signal peptide, a MalE signal peptide, a MglB signal peptide, a OmpA signal peptide, or a PelB signal peptide.

A signal peptide may be a yeast signal peptide. For example, the yeast signal peptide may be a SUC2 signal peptide or a PH05 signal peptide.

A signal peptide may be a mammalian signal peptide. For example, the mammalian signal peptide may a BM-40 signal peptide, a VSVG signal peptide, a chymotrypsinogen signal peptide, an interleukin-2 (IL-2) signal peptide, a Gaussia lucif erase signal peptide, a human serum albumin signal peptide, an influenza haemagglutinin signal peptide, or an insulin signal peptide.

Immobilization

The term "immobilization" refers to various methods and techniques to link proteins to supports. For example, immobilization can serve to stabilize the proteins so that its activity is not reduced or adversely modified by biological, chemical or physical exposure, especially during storage or in single-batch use.

Solid support

The term "solid support" refers to any solid (flexible or rigid) substrate onto which one or more compounds may be applied. For example, the solid support may be in the form of a bead, column, membrane, microwell or centrifuge tube. The solid support may be a bead and wherein the bead is a glass bead, or microbead, magnetic bead, or paramagnetic bead.

Cell

The term "cell" refers to any prokaryotic or eukaryotic cell. Examples of suitable prokaryotic cells include, for example, strains of E. coli (e.g., BL21, DH5a, XL-l-Blue, JM105, JM110, and Rosetta), Bacillus subtilis, Salmonella sp., and Agrobacterium tumefaciens. The cell may be a plant cell, a yeast, an insect or a mammalian cell. Examples of suitable mammalian cells include cell lines, such as, for example, human GM12878, K562, HI human embryonic, Hela, HUVEC, HEPG2, HEK-293, H9, MCF7, and Jurkat cells, mouse NIH-3T3, C127, and L cells, simian COSl and COS7 cells, quail QCl-3 cells, and Chinese hamster ovary (CHO) cells. The mammalian cells may be hematopoietic cells, neural cells, mesenchymal cells, cutaneous cells, mucosal cells, stromal cells, muscle spleen cells, reticuloendothelial cells, epithelial cells, endothelial cells, hepatic cells, kidney cells, gastrointestinal cells, pulmonary cells, T- cells.

A cell may be a cultured cell, e.g. in vitro or ex vivo. For example, cells cultured in vitro in a culture medium and environmental stimuli may be added to the culture medium. Alternatively, for ex vivo cultured cells, cells may be previously obtained from a subject. Cells can be obtained by biopsy or other surgical means known to those skilled in the art.

Protein synthesis

Any protein of the present disclosure may be synthesized using a chemical method known to the skilled artisan. For example, synthetic protein are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Protein production

Any protein of the present disclosure may be expressed by recombinant means. For example, the nucleic acid encoding the binding protein may be placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in cellular system or organism.

Typical promoters suitable for expression in bacterial cells include, for example, the lacz promoter, the Ipp promoter, temperature-sensitive _L or _R promoters, T7 promoter, T3 promoter, SP6 promoter or semi- artificial promoters such as the IPTG- inducible tac promoter or lacUV5 promoter. A number of other gene construct systems for expressing the nucleic acid fragment of the invention in bacterial cells are well- known in the art and are described, for example, in Ausubel et al. (1988), and Sambrook et al. (2001).

Numerous expression vectors for expression of recombinant polypeptides in bacterial cells have been described, and include, for example, PKC3, pKK173-3, pET28, the pCR vector suite (Invitrogen), pGEM-T Easy vectors (Promega), the pL expression vector suite (Invitrogen) or pBAD/thio— TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen), amongst others. Typical promoters suitable for expression in yeast cells such as, for example, a yeast cell selected from the group comprising Pichia pastoris, S. cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

Expression vectors for expression in yeast cells are preferred and include, for example, the pACT vector (Clontech), the pDBleu-X vector, the pPIC vector suite (Invitrogen), the pGAPZ vector suite (Invitrogen), the pHYB vector (Invitrogen), the pYD 1 vector (Invitrogen), and the pNMT 1, pNMT41, pNMT81 TOPO vectors (Invitrogen), the pPC86-Y vector (Invitrogen), the pRH series of vectors (Invitrogen), pYESTrp series of vectors (Invitrogen).

Preferred vectors for expression in mammalian cells include, for example, the pcDNA vector suite (Invitrogen), the pTARGET series of vectors (Promega), and the pSV vector suite (Promega).

Suitable methods for transforming and transfecting host cells can be found in

Sambrook et al. 2001 and other laboratory textbooks. In one example, nucleic acid may be introduced into prokaryotic cells using for example, electroporation or calcium- chloride mediated transformation. In another example, nucleic acid may be introduced into mammalian cells using, for example, microinjection, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, transfection mediated by liposomes such as by using Lipofectamine (Invitrogen) and/or cellfectin (Invitrogen), PEG mediated DNA uptake, electroporation, transduction by Adenoviuses, Herpesviruses, Togaviruses or Retroviruses and microparticle bombardment such as by using DNA-coated tungsten or gold particles. Alternatively, nucleic acid may be introduced into yeast cells using conventional techniques such as, for example, electroporation, and PEG mediated transformation.

Protein isolation

Following production/expression/synthesis, any protein of the present disclosure can be purified using a method known in the art. For example, affinity purification may be used to purify any protein of the present disclosure Methods for isolating a protein using affinity chromatography are known in the art and described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).

The degree of purity any protein of the present disclosure may be determined by various methods, including identification of a major large peak on HPLC OR UPLC. Methods for identifying a molecule that specifically binds with a peptide

The present disclosure provides for methods for identifying a molecule that specifically binds with a peptide e.g. a peptide contained within any modified binding protein described herein or a peptide contained within any complex described herein. As used herein, the term "specifically binds" or "binds specifically" means that a peptide of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a molecule or cell expressing the same than it does with alternative molecules or cells. For example, a peptide that specifically binds to a molecule binds to that molecule with greater affinity, avidity, more readily, and/or with greater duration than it binds to other molecules. It is also understood by reading this definition that, for example, a peptide that specifically binds to a first molecule may or may not specifically bind to a second molecule. As such, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another molecule, this is meant by the term "selective binding". In one example, "specific binding" of a peptide of the disclosure to a molecule, means that the peptide binds to the molecule with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, for example, 20 nM or less, such as, 15 nM or less or 10 nM or less or 5 nM or less or 1 nM or less or 500 pM or less or 400 pM or less or 300 pM or less or 200 pM or less or 100 pM or less.

Methods for detecting an interaction between a molecule and a peptide are well known in the art.

An interaction between a molecule and a peptide may be identified using affinity purification. Affinity purification techniques are known in the art and are described in, for example, Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Methods of affinity purification typically involve contacting a peptide with a specific target molecule, e.g., a target protein and, following washing to remove unbound or non- specifically bound peptides, eluting those peptides that remain bound to the target protein. By performing increasingly stringent washes, peptides having higher affinity for the target molecule are identified.

An interaction between a molecule and a peptide may be identified using a protein chip or series of pins having immobilized thereon a target may be contacted with a peptide. Preferably, the peptide is labelled with a detectable marker, e.g., a fluorescent marker. Following washing to remove any unbound peptide, the location of bound label is detected. The location of bound label is indicative of a peptide capable of binding to the target molecule. The identity of the peptide may then be conformed, e.g., using a method described herein, e.g., MALD-TOF. An interaction between a molecule and a peptide may be identified using a surface-plasmon resonance assay, such as, for example, Biacore sensor chip technology (Biacore AB, UK). The Biacore sensor chip is a glass surface coated with a thin layer of gold modified with carboxymethylated dextran, to which a target molecule is covalently attached. A peptide is then brought into contact with the target molecule. Essentially, a surface plasmon resonance assay detects changes in the mass of the aqueous layer close to the chip surface, through measuring changes in the refractive index. Accordingly, when a peptide binds to the target protein or nucleic acid the refractive index increases.

An interaction between a molecule and a peptide may be identified with a biosensor based on the detection of diffractive optics technology (light-scattering). Such biosensors are available commercially, e.g., from Axela Biosensors Inc., Toronto, Canada. Alternatively a biosensor may be used which is based on acoustic resonance, such as that produced by Akubio, Cambridge UK.

An interaction between a molecule and a peptide may be identified using a commercially available chip. For example, a GPCR chip may be used to identify an interaction between a peptide and a G-protein coupled receptor as described in Fang et al., 2002.

An interaction between a molecule and a peptide may be identified using a screen, such as, for example, a radioimmunoassay (RIA), an enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology. Such methods are known in the art and/or described herein.

An interaction between a molecule and a peptide may be identified using the two-hybrid assay. Modified two-hybird assays may be used including, for example, a PolIII two hybrid system, the Tribrid system, a ubiquitin based split protein sensor system, a Sos recruitment system as described in Vidal and Legrain 1999 or the three hybrid assay.

An interaction between a molecule and a peptide may be identified using biopanning. In one example, biopanning may involve a negative section step wherein peptides are incubated with a non-target population of cells in medium for a period of time and under conditions sufficient to allow peptides to adhere to the cells. Subsequent removal of the non-target population of cells from the medium will result in a proportion of peptides which have adhered to the cells being removed e.g., this negative selection may remove peptides with an affinity for adhering to a broad range of cells. In another example, biopanning may involve a positive section step wherein peptides from the one or more negative selection steps are incubated with a desirable target population of cells. Incubation of those peptides and cells for a period of time and under conditions sufficient to allow peptides to adhere to the target population of cells thereby isolating cell- specific or cell selective peptides.

Methods for directing a molecule to a target

The present disclosure provides a method for directing a molecule to a target comprising:

i) contacting a cell with a modified binding protein linked to a molecule {i.e. a conjugate or fusion protein), wherein the cell expresses a target linked to a protein binding partner, and wherein the binding protein forms a covalent isopeptide bond with the protein binding partner such that the molecule is directed to the target.

The contacting at step i) may be for a time and under conditions sufficient for the conjugate to enter a cell. Thus, it is within the scope of the disclosure that the peptide inserted into a region of the binding protein may be a cell penetrating peptide.

The method may additionally comprise determining or identifying that a conjugate has been directed to a target.

The method may also comprise producing cells that are stably or transiently transformed with a vector encoding the target linked to the protein binding partner.

Methods for identifying a peptide capable of translocating a membrane of a cell

The present disclosure provides a method of identifying a peptide capable of translocating a membrane of a cell comprising:

i) contacting a cell with a modified binding protein {i.e. a binding protein wherein a peptide is inserted into a region of the binding protein); and

ii) detecting the peptide in the cell.

A modified binding protein may be provided as a conjugate. For example, the conjugate may comprise a modified binding protein and a detectable label. In another example, a modified binding protein may be displayed on a particle.

The contacting at step i) may be for a time and under conditions sufficient for the modified binding protein to enter the endosome of the cell. In this example, the method may comprise an incubating at step after step ii) may be for a time and under conditions such that the modified binding protein is translocated out of the endosome. The method may additionally comprise treating the cell at step i) to remove modified binding protein that are associated with the membrane of the cells without disrupting the cell membranes. By "associated with the membrane" it is meant that the peptide is in physical relation with the cell other than by means of a mechanism that is capable of transporting the peptide through the membrane of that particular cell or internalizing the peptide in that particular cell. For example, treating the cells may comprise incubating the cells with a protease for a time and under conditions sufficient to remove and/or inactivate extrinsic modified binding proteins to the cells without disrupting the cell membrane.

The invention will now be further described with reference to the following, non-limiting examples.

Example 1: General Materials & Methods

General constructs

A FITC labelled binding partner (FITC-Partner) and a FITC labelled binding partner comprising an aspartic acid to alanine mutation (FITC-Partner ^DA ) are synthesised using the mild Fmoc chemistry method. The amino acid sequence for FITC-Partner is set forth in SEQ ID NO: 64. The amino acid sequence for FITC- Partner^ is set forth in SEQ ID NO: 83.

A biotin labelled binding partner (Bio-Partner) is synthesised using the mild

Fmoc chemistry method. The amino acid for Bio-Partner is set forth in SEQ ID NO: 65. Protein expression and purification

Recombinant proteins are expressed in E. coli BL21 (DE3). LB media supplemented with 0.5 mg/mL kanamycin was inoculated from a glycerol stock. Overnight cultures are diluted 1000-fold, grown in auto-induction media (Sigma) with 0.5 mg/mL kanamycin and then cultured at 37 °C overnight.

After overnight growth at 37 °C with agitation, 1 1000th of the primary culture is used to inoculate autoinduction media (Sigma) supplemented with 0.5 mg/mL kanamycin. After overnight growth at 37 °C with agitation, cells are harvested at 4,000 rpm for 20 min and resuspended in Bug Buster Master Mix (Novagen) a protease inhibitor cocktail tablet. Solutions were incubated at room temperature for 30 min and then pelleted at 4°C.

Metal-affinity chromatography (Ni-NTA, Qiagen) is used to affinity purify each sample that contains a hexahistidine tag. All samples are desalted using PD-10 desalting columns (GE Life Sciences) as per manufacturer's instructions. Protein concentration and purity is determined using the Pierce BCA analysis (Thermo Fisher Scientific) and SDS-PAGE analysis as previously described (Sambrook et al, 1989). SDS-PAGE

SDS-PAGE is performed on 10 to 20% gradient gels. Dithiothreitol (DTT, Sigma) is added to a final concentration of 100 mM. Samples are then mixed with 6x SDS-PAGE loading buffer (0.23 M Tris-HCl, 0.24 % glycerol, 6.7 % SDS and 12 mM bromophenol blue). Samples are heated at 95 °C for 7 min before loading onto the gel. Gels were stained with Coomassie Blue, imaged using a Bio-Rad ChemiDoc XRS+, and analyzed using Image Lab 3.0 software (Bio-Rad).

Mass spectrometry

The presence or absence of an isopeptide bond formation is determined using LC/MS. All samples are desalted using PD-10 desalting columns (GE Life Sciences) as per manufacturer's instructions.

Display on bacteriophage

For T phage, vector construct designated, T7-PreScission-Avi, was generated for mid-copy number display of proteins using T7 Select 10 vector (Novagen) as a template. The T7-PreScission-Avi vector encodes a Rhinovirus 3C protease recognition site and an Avi-Tag and. The vector T7-PreScission-Avi comprises an EcoRI and Sail site positioned 3' of the nucleic acid encoding a 10B capsid protein to provide for insertion of nucleic acid encoding the desired cassette. Cassettes from pET28a+ vectors are amplified using specific primers that bind to the Mfel and Sail restriction sites. Amplified fragments are column purified, digested with Mfel/Sall and inserted in the T7-PreScission-Avi vector cut with EcoRI/Sall. Specific methods for cloning, propagation and maintenance are used as specified in the manual supplied with the T7Select Packaging Kit (Merck Millipore).

For filamentous phage, vector construct designated, pNp3, was generated. pNp3 is an M13 vector comprising nucleic acid encoding a PelB leader, a hexahistidine tag, hemagglutinin tag, and a M13 pill coat protein. The vector pNp3 comprises an EcoRI and Sail site positioned 3' of the nucleic acid encoding the M13 pill coat protein to provide for insertion of nucleic acid encoding the desired cassette. pNp3 also contains a frameshift base between the EcoRI and Sail site that shifts the reading frame to ablate correct translation of pill when no DNA is inserted into the multiple cloning site. This avoids expression of wild-type phage that do not display a protein. Cassettes from pET28a+ vectors are amplified using specific primers that bind to the Mfel and Sail restriction sites. Amplified fragments are column purified, digested with Mfel/Sall and inserted in the pNp3 cut with EcoRI/ Sail.

Generation of binding protein (BP) cassette and modified binding protein (MP) cassettes for use in bacteriophage display

For the display of a nucleic acid fragment library between a binding protein and a binding protein partner, a BP library cassette was generated using pET28+ as a template. The BP library cassette comprises an EcoRI restriction site between nucleic acid encoding the binding protein and nucleic acid encoding binding protein that includes a frameshift base that shifts the reading frame to ablate correct translation of the binding protein partner when no DNA is inserted into the EcoRI restriction site. The nucleic acid sequence of the BP library cassette is set forth in SEQ ID NO: 90.

For the display of a nucleic acid fragment library in any of the designated regions of the modified binding protein, several MB library cassettes are generated using pET28+ as a template. Briefly, the MP library cassette with an EcoRI restriction site in the designated loop region that includes a frameshift base that shifts the reading frame to ablate correct translation of modified binding protein 3' to the EcoRI cloning site when no DNA is inserted. The nucleic acid sequence of a MP library cassette is set forth in SEQ ID NO: 91.

To create a T7 display vector, the BP library cassette and MB library cassettes are cloned into T7-Prescission-Avi cut with EcoRI/ Sail. The resulting vectors are designated, T7-BP-Entry and T7-MP-Entry.

To create a M13 display vector, the BP library cassette and MB library cassettes are cloned into the pNp3 cut with EcoRI/ Sail. The resulting vectors are designated, M13-BP-Entry and M13-MP-Entry.

Bacteriophage display library

A highly diverse mixture of nucleic acids is produced from coding and non- coding regions of viral, prokaryotic and eukaryotic genomes, essentially as described in US Pat. No. 7,270,969. Nucleic acid fragments are generated using multiple consecutive rounds of PCR using tagged random oligonucleotides and mixture of nucleic acid fragments produced from viral, prokaryotic and eukaryotic genomes were digested with the restriction endonuclease Mfel, purified e.g., using a QIAquick PCR purification column (QIAGEN) as per manufacturer's instructions, and retained for ligation into a compatible EcoRI site of T7 vector or a M13 vector for subsequent display.

Nucleic acid fragment libraries for T7 bacteriophage are generated by inserting Mfel digested nucleic acid fragments in T7-BP-Entry cut with EcoRI or T7-MP-Entry cut with EcoRI. T7 bacteriophage are prepared as specified in the manual supplied with the T7 Select Packaging Kit (Merck Millipore).

Nucleic acid fragment libraries for M13 bacteriophage are generated by inserting Mfel digested nucleic acid fragments in M13-BP-Entry cut with EcoRI or cut with EcoRI. M13 bacteriophage are prepared using standard methods.

Bacteriophage labelling

PEG precipitated T7 and M13 phage are labelled with either AlexaFluor 488 (AlexaFluor® 488 carboxylic acid 2,3,5, 6-tetrafluorophenyl ester 5-isomer) or Oregon Green 488 (Oregon Green® 488 carboxylic acid, succinimidyl ester 5- isomer). Approximately 10 ¹² phage particles are incubated with 50μ of fluorescent dye followed by purification from un-reacted dye by triple PEG precipitation or size exclusion chromatography (SEC, S200-HR). The number of dye molecules per phage particle is calculated using Beer- Lambert's Law. Example 2: Display of a single-chain protein

Bovine pancreatic trypsin inhibitor (BPTI) is a well-characterised single-chain protein which contains three disulfide bonds, an alpha helix, a short 3-helix and a 3- stranded beta sheet. BPTI contains 58 amino acid residues and inhibits the proteolytic activity of trypsin. The amino acid sequence of BPTI is set forth in SEQ ID NO: 23.

Materials and Methods

Constructs

pET28a+ partner-BPTI-binding protein (PBB), pET28a+ binding protein-BPTI- partner (BBP), pET28a+ binding protein-BPTI ^m - partner (BB ^m P), pET28a binding protein-BPTI-partner ^DA (BBP ^DA ), pET28a+ binding protein-BPTI ^m -partner ^DA (BB ^m P ^DA ), and pET28a+ modified binding protein-BPTI (MB) were with codons optimised for expression in Escherichia coli (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes were cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 1. Protein expression and purification

BBP, BB ^m P, BBP ^DA , BB ^m P ^DA and MB were expressed and purified using the method described in Example 1. Complex formation

To test the ability of MB to interact with a protein binding partner, MB was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio. In one example, MB was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction was then stopped.

The reaction mixture was analysed by SDS PAGE under low light conditions.

The gels were also then read under a FITC channel. Gels are also stained with Coomassie blue stain. The efficiency of the interactions between the MB and FITC- Partner was determined by comparing the molecular mass of the MB alone or FITC- Partner alone with the molecular mass of a sample of the reaction mixture comprising MB and FITC-Partner. In addition, densitometry was used to evaluate conjugation reaction efficiency.

Display ofBPTI on bacteriophage

PBB, BBP, BB ^m P, BBP ^DA , BB ^m P ^DA and MB were displayed on T phage using the method described in Example 1.

Trypsin inhibition assay

To test that BPTI is function when expressed as PBB, BBP, BB ^m P, BBP ^DA , BB ^m P ^D and MB, the ability of the recombinant proteins to inhibit the proteolytic activity of trypsin is performed using a colour-metric QuantiCleave protease assay kit (Pierce) as per manufacturer's instructions. Briefly, in a microtitre plate 50 ul serial dilutions of trypsin (l-in-2 from 0.5 mg/ml to 3.9 x 10 ^" mg/ml) were incubated with 100 ul succinylated casein in a 50 mM borate buffer pH 8.5, and 50 ul of purified PBB, BBP, BBmP, BBP DA, BBmPD and MB proteins for 30 minutes at room temperature. Blanks of every sample were measured using 100 ul assay buffer instead of succinylated casein. Protease activity is measured as A450 using a fluorescence microplate reader 20 min after the addition of chromogenic reagent 2,4,6- trinitrobenzene sulfonic acid, which reacts with the primary amine of digested peptide and produces the colour reaction that can be quantified by the absorbance reader. Results

Complex Formation

When incubated with FITC-partner, BBP did not form a complex as evidenced by the presence of a single band corresponding to the unconjugated mass of BBP at in the Coomassie Blue stained SDS PAGE gel (~21kDa), and the absence of a fluorescent band at the expected mass of BBP-FITC-partner complex in the FITC channel (~25kDa). Together these data indicate that the binding protein and protein binding partner, separated by BPTI, formed a stable, irreversible conjugate for BBP and that BPTI is thus cyclised.

PBB did bind a small amount of FITC-partner as evidenced by a faint fluorescent band at the expected molecular weight for a PBB-FITC-partner conjugate (~27kDa) and a large band at ~3.5kDa corresponding to unreacted FITC-partner in the FITC channel analysed SDS PAGE gel. The faint band corresponded to a conjugation efficiency of less than 2% by densitometry. The Coomassie Blue stain SDS PAGE gel also displayed a faint band at ~27kDa, but this was less than 1% of the large band at ~24kDa corresponding to the unconjugated PBB. These data indicate that the majority of PBB is unable to ligate with FITC-partner, and that the protein binding partner and binding protein, separated by BPTI, form a stable conjugate.

BBP ^DA was able to form a conjugate with FITC-partner as evidenced by a fluorescent band at ~25kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the BBP ^DA -FITC-partner conjugate. This indicates that the BBP ^DA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner and that BPTI is thus not cyclised.

MB formed a conjugate with FITC-partner as evidenced by a band at the correct molecular weight for the MB-FITC-partner conjugate in both FITC channel and Coomassie stained SDS gels (~24kDa). The efficiency of conjugation was determined by densitometry to be -70%, which was similar to that expected of the binding protein. These data indicate that the modified binding protein folds correctly when BPTI is included in the modified site, is able to efficiently conjugate with FITC-partner and that BPTI is cyclised.

T7 bacteriophage displayed BBP did not form a conjugate when incubated with FITC-partner, as evidenced by the absence of any fluorescent bands in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BBP was unable to conjugate with FITC-partner and is thus cyclised. T7 bacteriophage displayed BBPDA when incubated with FITC-partner, formed a conjugate as evidenced by the presence of a fluorescent band at the correct molecular weight for the T7-capsid-displayed-BBPDA-FITC-partner conjugate (~63kDa) in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BBPDA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner.

T7 bacteriophage displayed MB formed a conjugate with FITC-partner as evidenced by a fluorescent band at the correct molecular weight (~62kDa) for the T7- capsid-displayed-MB-FITC-partner conjugate in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed modified binding protein folds correctly when BPTI is included in the modified site, and is able to efficiently conjugate with FITC-partner, and that the BPTI is cyclised.

Trypsin inhibition assay

To determine whether a BPTI domain inserted into a region of a binding protein or a BPTI domain linked at a first end to a binding protein and at a second end to a protein binding partner were correctly folded and functional, the ability of PBB, BBP and MB to inhibit tryspin activity was examined. .

As shown in Figure 1, reduced absorbance was observed on the addition of PBB or BBP. This reduced absorbance is indicative that BPTI when linked at a first end to a binding protein and at a second end to a protein binding partner is correctly folded. Furthermore, these results indicate that BPTI which is displayed in this matter is able to specifically bind to trypsin and to inhibit the proteolytic activity of trypsin.

As shown in Figure 2, reduced absorbance was observed on the addition of MB. No change in absorbance was observed on the addition of MS which contains a SH3 domain instead of a BPTI domain. This reduced absorbance is indicative that BPTI when inserted into a region of a binding protein is correctly folded. Furthermore, these results indicate that BPTI which is displayed in this matter is able to specifically bind to trypsin and to inhibit the proteolytic activity of trypsin. Table 1

Single-chain protein cassettes

Cassette Description Amino Acid

Sequence

Partner-BPTI-binding This cassette comprises a protein binding SEQ ID NO: 25 protein (PBB) partner, a bovine pancreatic trypsin

inhibitor, and a binding protein.

Binding protein-BPTI- This cassette comprises a binding protein, a SEQ ID NO: 26 partner (BBP) bovine pancreatic trypsin inhibitor, and a

protein binding partner.

Binding protein- This cassette comprises a binding protein a SEQ ID NO: 27 ΒΡΤ -partner (BB ^m P) bovine pancreatic trypsin inhibitor wherein

all cysteine residues have been replaced by

serine residues and a protein binding partner.

Binding protein-BPTI- This cassette comprises a binding protein, a SEQ ID NO: 28 partner ^m (BBP ^DA ) bovine pancreatic trypsin inhibitor and a

modified protein binding partner wherein

the aspartic acid residue capable of forming

a covalent isopeptide bond is replaced with

an alanine residue.

Binding protein- This cassette comprises a binding protein, a SEQ ID NO: 29 BPTI ^m -partner ^m bovine pancreatic trypsin inhibitor wherein

(BB ^m P ^DA ) all cysteine residues have been replaced by

serine residues and a modified protein binding partner wherein the aspartic acid

residue capable of forming a covalent isopeptide bond is replaced with an alanine

residue.

Modified binding This cassette comprises a modified binding SEQ ID NO: 30 protein-BPTI (MB) protein wherein a bovine pancreatic trypsin

inhibitor is inserted into a region of the modified binding protein. Example 3: Display of a non-catalytic domain

The SRC Homology 3 domain (SH3) is a noncatalytic domain. The basic fold of SH3 domains contain five anti-parallel beta-strands packed to form two perpendicular beta-sheets. SH3 contains 58 amino acid residues. SH3 does not contain any cysteine residues. The amino acid sequence of SH3 is set forth in SEQ ID NO: 31.

Materials and Methods

Constructs

pET28a+ partner-SH3-binding protein (PSB), pET28a+ SH3-binding protein (SB), pET28a+ binding protein- SH3 -partner (BSP), pET28a+ binding protein-SH3 (BS), pET28a+ binding protein-SH3-partner ^DA (BSP ^DA ) and pET28a+ modified binding protein-SH3 (MS) are synthesized with codons optimised for expression in Escherichia coli (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes are cloned into the NcoIZ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 2.

Protein expression and purification

PSB, SB, BSP, BS, BSP ^DA and MS are expressed and purified using the method described in Example 1.

Complex formation

To test the ability of MS to interact with a protein binding partner, MS was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio. In one example, MS was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction is then stopped.

The reaction mixture is analysed by SDS PAGE under low light conditions. The gels are then read under a FITC channel. Gels are also stained with Coomassie blue stain. The efficiency of the interactions between the MS and FITC-Partner was determined by comparing the molecular mass of the MS alone or FITC-Partner alone with the molecular mass of a sample of the reaction mixture comprising MB and FITC- Partner. In addition, densitometry was used to evaluate reaction efficiency.

Display ofSH3 on bacteriophage

PSB, SB, BSP, BS, BSP ^DA and MS were displayed on T phage using the method described in Example 1. Table 2

Non-catalytic domain cassettes

Cassette Description Amino Acid

Sequence

Partner-SH3- This cassette comprises a protein binding partner, a SEQ ID NO: 32 Binding protein SRC Homology 3 Domain and binding protein.

(PSB)

SH3-Binding This cassette comprises a SRC Homology 3 Domain SEQ ID NO: 33 protein (SB) and a binding protein.

Binding protein- This cassette comprises a binding protein, a SRC SEQ ID NO: 34 SH3-Partner Homology 3 Domain and a protein binding partner.

(BSP)

Binding protein- This cassette comprises a binding protein and a SRC SEQ ID NO: 35 SH3 (BS) Homology 3 Domain.

Binding protein- This cassette comprises a binding protein, a SRC SEQ ID NO: 36

SH3-Partner ,DA Homology 3 Domain and a protein binding partner

(BSP ^DA ) wherein the aspartic acid residue capable of forming

a covalent isopeptide bond is replaced with an

alanine residue.

Modified binding This cassette comprises a modified isopeptide protein SEQ ID NO: 37 protein-SH3 (MS) wherein a SRC Homology 3 Domain is inserted into

a region of the modified binding protein.

Results

Complex formation

When incubated with FITC-partner, BSP did not form a conjugate as evidenced by the presence of a single band corresponding to the unconjugated mass of BSP at in the Coomassie Blue stained SDS PAGE gel (~21kDa), and the absence of a fluorescent band at the expected mass of BSP-FITC-partner conjugate in the FITC channel analysed SDS PAGE gel (~25kDa). Together these data indicate that the binding protein and protein binding partner, separated by SH3, formed a stable, irreversible conjugate for BSP. PSB did bind a small amount of FITC-partner upon incubation, as evidenced by a faint fluorescent band at the expected molecular weight for a PBB-FITC-partner conjugate (~28kDa) in the FITC channel analysed SDS PAGE gel, however this corresponded to a conjugation efficiency of less than 1% by densitometry. The Coomassie Blue stain SDS PAGE gel also displayed a faint band at ~28kDa, but again this was less than 1% of the large band at ~24kDa corresponding to the unconjugated PSB. These data indicate that the majority of PSB is unable to conjugate with FITC- partner, and that the protein binding partner and binding protein, separated by SH3, form a stable conjugate.

BSP ^DA was able to form a conjugate with FITC-partner as evidenced by a fluorescent band at ~25kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the BSP ^DA -FITC-partner conjugate.

MS formed a conjugate with FITC-partner as evidenced by a band at the correct molecular weight for the MB -FITC-partner conjugate in both FITC channel analysed and Coomassie stained SDS PAGE gels (-24 kDa). The efficiency of conjugation was determined by densitometry to be 60%, and similar to that expected of the binding protein. These data indicate that the modified binding protein folds correctly when SH3 is included in the modified site, and is able to efficiently conjugate with FITC-partner.

T7 bacteriophage displayed BSP did not form a conjugate when incubated with FITC-partner, as evidenced by the absence of any fluorescent bands in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BSP was unable to conjugate with FITC-partner and that the SH3 domain is thus cyclised.

T7 bacteriophage displayed BSP ^DA when incubated with FITC-partner, formed a conjugate with the presence of a fluorescent band at the correct molecular weight for the T7-capsid-displayed-BSP ^DA -FITC-partner conjugate (~63kDa) in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BSP ^DA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner.

T7 bacteriophage displayed MS formed a conjugate with FITC-partner as evidenced by a fluorescent band at the correct molecular weight (-62 kDa) for the T7- capsid-displayed-MS-FITC-partner conjugate in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed modified binding protein folds correctly when SH3 is included in the modified site, and is able to efficiently conjugate with FITC-partner, and that the SH3 is cyclised. Example 4: Display of a peptide mimetic

p53 is best known as a tumor suppressor that transcriptionally regulates, in response to cellular stresses such as DNA damage or oncogene activation, the expression of various target genes that mediate cell-cycle arrest, DNA repair, senescence or apoptosis. The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53. Several peptide inhibitors of the p53-MDM2/MDMX interactions have been identified including p53 ^17"28 , PMI, PMI ^N8A and PDI (see for example Hu et al. 2007, Li et al. 2010 and Ji et al. 2013). The amino acid sequence of p53 17 ^" 28 is set forth in SEQ ID NO: 38. The amino acid sequence of PMI is set forth in SEQ ID NO: 39. The amino acid sequence of PMI ^N8A is set forth in SEQ ID NO: 40. The amino acid sequence of PDI is set forth in SEQ ID NO: 41.

COP1, the constitutive morphogenic protein 1 is an E3 ubiquitin ligase that also regulates the activity of the tumour suppressor protein p53 by binding to its DNA- binding domain. A peptide inhibitor of the p53-COPl interaction has been identified (see for example Yamada et al. 2013 and, Yamada, Das Gupta and Beattie 2013). The amino acid of p28 is set forth in SEQ ID NO: 100.

To assess whether the p53-MDM2/ MDMX and p53-COPl peptide inhibitors were amenable to structural constraint within a scaffold loop, p53 ^17"28 , PMI ^N8A , PDI and p28 sequences were inserted into a loop within the modified binding protein

(Figure 3), generating the constructs modified binding protein p53 17 ^" 28 (Mp5317 ^" 28 ), modified binding protein PMI ^N8A (MPMI ^N8A ), modified binding protein PDI (MPDI) and modified binding protein p28 (Mp28).

To compare the impact of structural constraint upon the p53-MDM2/ MDMX and p53-COPl peptide inhibitors, unconstrained variants were also constructed. The p53 ^17"28 , PMI ^N8A and p28 peptide sequences were added to the C-terminus of the binding protein (Figure 3), generating binding protein p53 17 ^" 28 (Bp5317 ^" 28 ), binding protein PMI ^N8A (BPMI ^N8A ) and binding protein p28 (Bp28).

The p53 transcription factor is located in the cell nucleus; to evaluate the biological action of the binding protein and modified binding protein p53 mimetics, they will need to be conjugated to a cell-penetrating peptide protein binding partner (CPP-partner). The CPP-component will facilitate the translocation of the binding protein and modified binding protein p53 mimetics from the extracellular space into the cytoplasm from where they can interact with either MDM2/ MDMX or COP1. Materials and Methods

Constructs

pET28a+ modified binding protein-p53 ^17-28 (Mp53 ^17"28 ), pET28a+ modified binding protein-PMI (MPMI), pET28a+ modified binding protein-PMI ^N8A (MPMI ^N8A ), pET28a+ modified binding protein-PDI (MPDI) and pET28a+ modified binding protein-p28 (Mp28) are synthesized (DNA 2.0, Menlo Park, CA, USA). pET28a+ binding protein-p53 ^17-28 (Bp53 ^17"28 ), pET28a+ binding protein-PMI ^N8A (BPMI ^N8A ) and pET28a+ binding protein-p28 (Bp28) are synthesized (DNA 2.0, Menlo Park, CA, USA).The synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 3.

Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector.

Protein expression and purification

Bp53 ^17"28 , BPMI ^N8A , Bp28, Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI and Mp28 are expressed in bacteria and purified using the method described in Example 1.

Complex formation

To test the ability of Mp53 ^17"18 , MPMI, MPMI ^N8A , MPDI and Mp28 to interact with a protein binding partner, each protein was mixed with CPP-Partner at a 1: 1 to 1.10 stoichiometric ratio. In one example, the protein was mixed with CPP-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction is then stopped.

The reaction mixture is analysed by Coomassie blue stained SDS PAGE gel.

The efficiency of the interactions between the Mp53 ^17"18 , MPMI, MPMI ^N8A , MPDI and Mp28 with CPP-Partner was determined by comparing the molecular mass of the Mp53 ^17"18 , MPMI, MPMI ^N8A , MPDI or Mp28 alone or CPP-Partner alone with the molecular mass of a sample of the reaction mixture comprising Mp53 17 ^" 18 , MPMI, MPMI ^N8A , MPDI and Mp28, and CPP-Partner. In addition, densitometry was used to evaluate reaction efficiency.

Cells

T47D breast cancer cell lines are obtained. T47D breast cancer cells express a mutated type of p53 which is localized in the cytoplasm and is known to be sensitive to MDM2 inhibition. HCT116 human colon cancer cell lines are obtained. As a control, HCT116's p53 deficient subline HCT116 p537- are obtained.

U87 glioma cell lines are obtained. U87 glioma cell lines express a mutated type of p53. As a control, U251 glioma cell lines are also obtained which express a wild type p53.

CHO-K1 cell lines are obtained. CHO-K1 cell lines express wild type p53. Cell viability assay

The activity of the Bp53 ^17"28 , BPMI ^N8A , Bp28, Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI, Mp28, Bp53 ^17"28 -CPP-partner conjugate, BPMI ^N8A -CPP-partner conjugate, Bp28-CPP-partner conjugate, Mp53 ^17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate, MPMI ^N8A -CPP-partner conjugate, MPDI-CPP-partner conjugate and Mp28- CPP-partner conjugate is evaluated in selected cells.

Cell viability following addition of Bp53 ^17"28 , BPMI ^N8A , Bp28, Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI, Mp28, Bp53 ^{17 28} -CPP-partner conjugate, BPMI ^N8A -CPP- partner conjugate, Bp28-CPP-partner conjugate, Mp53 17 ^" 28 -CPP-partner conjugate, MPMI-CPP-partner conjugate , MPMI ^N8A -CPP-partner conjugate, MPDI-CPP-partner conjugate and Mp28-CPP-partner conjugate is evaluated using the PrestoBlue® Assay (LifeTech). Briefly, cells are seeded into 96 well plates and allowed to settle overnight. Bp53 ^17"28 , BPMI ^N8A , Bp28, Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI, Mp28, Bp53 ^17"28 - CPP-partner conjugate, BPMI ^N8A -CPP-partner conjugate, Bp28-CPP-partner conjugate, Mp53 ^17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate , MPMI ^N8A -CPP- partner conjugate, MPDI-CPP-partner conjugate and Mp28-CPP-partner conjugate are added to cells at a range of concentration in complete medium. At 24 and 48 h time points a 1/lOth volume of the PrestoBlue® reagent is be added to the media and the cells are incubated for a further 30 min. Fluorescent signal indicating cell metabolic activity are then be measured as per manufacturer's instructions using an EnSpire® multimode plate reader (Perkin Elmer).

In the cases for constructs containing p53 ^17"28 , PMI, PMI ^N8A and PDI peptide mimetics, namely Bp53 ^17"28 , BPMI ^N8A , Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI, Bp53 ^17"28 - CPP-partner conjugate, BPMI ^N8A -CPP-partner conjugate, Mp53 ^17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate , MPMI ^N8A -CPP-partner conjugate, and MPDI-CPP-partner conjugate, increased cell death indicates inhibition of the p53- MDM2/MDMX interaction. Increased cell death also indicates restoration of the normal programmed cell death pathways mediated by the P53 tumour suppressor. In the case for constructs containing the p28 peptide mimetic, namely Bp28, Mp28, Bp28-CPP-partner conjugate and Mp28-CPP-partner conjugate, increased cell death indicates inhibition of the p53-COPl interaction, increased levels of p53 and restoration of the normal programmed cell death pathways mediated by the P53 tumour suppressor pathway.

Results

Complex formation

Bp53 17 ^" 28 forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band on a Coomassie Blue stained SDS PAGE gel corresponding to the molecular weight of the Bp53 17 ^" 28 -CPP-partner conjugate

(~19.5kDa) in comparison the molecular weight of the unconjugated Bp53 17 28 (~14kDa). Densitometry measurements indicated that the conjugation efficiency for the reaction of Bp53 ^17-28 with CPP-partner was 74% (Figure 5).

BPMI ^N8A forms a conjugate with CPP-partner when incubated together, as evidenced by a band at ~19.5kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the BPMI ^N8A -CPP-partner conjugate in comparison to the unconjugated BPMI ^N8A molecular weight of ~14kDa. Densitometry measurements indicted that the conjugation efficiency for the reactions of BPMI ^N8A with CPP-partner was 60%. (Figure 5).

Bp28 forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ~21kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the Bp28 -CPP-partner conjugate in comparison to the unconjugated Bp28 molecular weight of ~15.5kDa. Densitometry measurements indicated that the conjugation efficiency for the reaction of Bp28 with CPP-partner was 43% (Figure 5).

Mp53 17 ^" 28 forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band at ~20.5kDa in a Coomassie Blue stained SDS PAGE gel. This molecular weight corresponds to the expected molecular weight for the

Mp53 17 ^" 28 -CPP-partner conjugate. The unconjugated Mp5317 ^" 28 molecular weight is ~15kDa. Densitometry measurements indicated that conjugation efficiency for the reaction of Mp53 17 ^" 28 with CPP-partner was 70% (Figure 4). This data indicates that the modified binding protein folds correctly when p53 17 28 is included in the modified site, and retains its ability to conjugate CPP-partner. MPMI forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ~20kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the MPMI ^N8A -CPP-partner conjugate in contrast to the unconjugated MPMI ^N8A molecular weight of ~15kDa. The conjugation efficiency of the reaction between MPMI ^N8A and CPP-partner was determined to be 67% by densitometry (Figure 4). These data indicate that the modified binding protein folds correctly when PMI ^N8A is included in the modified site, and retains its ability to conjugate CPP-partner.

MPDI forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band at ~20.5kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the MPDI-CPP- partner conjugate, in comparison to the unconjugated MPDI molecular weight of ~15kDa. The conjugation efficiency of the reaction between MPDI and CPP-partner was determined to be 34% by densitometry (Figure 4). These data indicate that the modified binding protein folds correctly when PDI is included in the modified site, and retains its ability to conjugate CPP-partner.

Mp28 forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ~22kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the Mp28 -CPP-partner conjugate, in comparison to the unconjugated Mp28 molecular weight of ~16.5kDa. The conjugation efficiency of the reaction between Mp28 and CPP-partner was determined to be 68% by densitometry (Figure 4). This data indicates that the modified binding protein folds correctly when p28 is included in the modified site, and retains its ability to conjugate CPP-partner.

Cell viability

To determine whether a p53 mimetic inserted into a region of a binding protein or a p53 mimetic linked at the C-terminal end of a binding protein were correctly folded, the ability of Bp53 ^17"28 , BPMI ^N8A , Mp53 ^17"28 , MPMI, MPMI ^N8A , MPDI, Bp53 ^{17" 28} -CPP-partner conjugate, BPMI ^N8A -CPP-partner conjugate, Mp53 ^17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate , MPMI ^N8A -CPP-partner conjugate, and MPDI-CPP-partner conjugate to inhibit MDM2/ MDMX or COP1 was evaluated in T47D breast cancer cells, where increased cell death indicates reinstatement of the P53 tumour suppressor pathway. CHO-K1 cells, with wild type P53 were evaluated in parallel as a negative control. As shown in Figure 6, in the absence of CPP-partner, Mp53 ^" does not inhibit cell viability in either T47D (A) or CHO-Kl (C) cell lines. The Mp53 ^17"28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner (B) but does not impact CHO-Kl cell viability (D). These data indicate that in p53-sensitive T47D, the cyclised Mp53 17 ^" 28 -CPP-conjugate is active.

As shown in Figure 7, in the absence of CPP-partner, both MPMI ^N8A and MPDI do not inhibit cell viability in T47D cells. The MPMI ^N8A -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner. The MPDI-CPP-partner conjugate also inhibits T47D cell viability in a dose dependent manner. These data indicate that in P53 sensitive T47D cells, both MPMI ^N8A -CPP-partner and MPDI-CPP- partner conjugates are active. (Constrained in modified binding protein, yet still active) In the absence of CPP-partner, Mp28 does not inhibit cell viability in either T47D or CHO-Kl cells (Figure 8). The Mp28-CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner, but does not impact on the cell viability of CHO-Kl cells. These data indicate that in p53 sensitive-T47D cells, cyclised Mp28- CPP-partner is biologically active.

In the absence of CPP-partner, Bp53 17 ^" 28 does not inhibit cell viability in either T47D (A) or CHO-Kl (C) cell lines (Figure 9). The Bp53 ^17"28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner (B) but does not impact CHO- Kl cell viability (D) (Figure 9). These data indicate that in p53 sensitive T47D, the non-cyclised Bp53 17 ^" 28 -CPP-conjugate is active.

In the absence of CPP-partner, BPMI ^N8A does not inhibit cell viability in T47D cells (Figure 10). The BPMI ^N8A -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner (Figure 10). These data indicate that in p53 sensitive T47D cells, non-cyclised BPMI ^N8A -CPP-partner is active.

In the absence of CPP-partner, Bp28 does not inhibit cell viability in either T47D or CHO-Kl cells (Figure 11). The Bp28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner, but does not impact on the cell viability of CHO-Kl cells (Figure 11). These data indicate that in p53 sensitive T47D cells, non- cyclised Bp28-CPP-partner is biologically active.

As a negative control, it was determined that the CPP-partner itself does not impact on the viability of either T47D or CHO-Kl cells, even at the highest concentration (Figure 12). This indicates that the CPP-partner is non-toxic. The PBS buffer also did not impact on the viability of either T47D or CHO-Kl cells, indicating it is non-toxic. Table 3

Peptide mimetic cassettes

Cassette Description Amino Acid

Sequence

Modified binding This cassette comprises a modified binding protein SEQ ID NO: 42 protein-p53 ^{17 28} wherein a p53 mimetic, p53 ^{17 28} , is inserted into a

(Mp53 ^17~28 ) region of the isopeptide protein.

Modified binding This cassette comprises a modified binding protein SEQ ID NO: 43 protein-PMI (MP) wherein a p53 mimetic, PMI, is inserted into a

region of the isopeptide protein.

Modified binding This cassette comprises a modified binding protein SEQ ID NO: 44 protein-PMI ^N8A wherein a p53 mimetic, PMI ^N8A , is inserted into a

(MPMI ^N8A ) region of the isopeptide protein.

Modified binding This cassette comprises a modified binding protein SEQ ID NO: 45 protein-PDI wherein a p53 mimetic, PDI, is inserted into a

(MPDI) region of the isopeptide protein.

Modified binding This cassette comprises a modified binding protein SEQ ID NO: protein-p28 (Mp28) wherein a p53 mimetic, p28, is inserted into a 126

region of the isopeptide protein

Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: p53 ^17"28 (Bp53 ^17~28 ) p53 mimetic, p53 ^{17 28} , is appended on the C- 127

terminus of the binding protein

Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: PMI ^N8A (BPMI ^N8A ) p53 mimetic, PMI ^N8A , is appended on the C- 128

terminus of the binding protein

Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: p28 (Bp28) p53 mimetic, p28, is appended on the C-terminus of 129

the binding protein Example 5: Display of a peptide mimotope

Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. Rituximab is known to interact with a 15 amino acid loop of CD20. A peptide mimotope of the CD20 epitope (PMCD20) has been shown to be recognized by Rituximab (Du et al. 2007). The amino acid sequence of PMCD20 is set forth in SEQ ID NO: 46. Rituximab does not bind to a mutated version of PMCD20 where the cysteine residues are substituted for serine residues (PMCD20 ^m ). The amino acid sequence of PMCD20 ^m is set forth in SEQ ID NO: 47. Materials and Methods

Constructs

pET28a+ binding protein-PMCD20-partner (BPP), pET28a+ binding protein- PMCD20 ^m -partner (BP ^m P), pET28a+ binding protein-PMCD20-partner ^DA (BPP ^DA ), pET28a+ binding protein- PMCD20 ^m - partner ^DA (BP ^m P ^DA ), pET28a+ modified binding protein-PMCD20 (MP) pET28a+ modified binding protein-PMCD20 ^m (MP ^m ) are synthesized (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 4.

Protein expression and purification

BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP and MP ^m are expressed and purified using the method described in Example 1. Display of peptide mimotopes on bacteriophage

BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP and MP ^m are displayed on T phage using the method described in Example 1.

ELISA assay

The ability of BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP or MP ^m to bind Rituximab is assessed using an ELISA assay. Briefly, Rituximab is coated onto 96-well polystyrene plates at approximately 2 ug/ml overnight at 4 °C. The plates are blocked with DELFIA assay buffer with PBS 1% BSA (v/v) at room temperature for 1 hour. Following washing of the blocking buffer (3x, PBS/ TBS 0.05% Tween-20), BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP or MP ^m are added and incubated for 1 hour at room temperature with shaking. Following washing (5x, PBS 0.05% Tween-20), a mouse anti-His antibody (1- in-2000 dilution in PBS) is added and incubated with shaking at room temperature for 1 hour. Following another round of washing (5x, PBS 0.05% Tween-20), an anti-mouse- HRP antibody (1 -in- 10000) is added and incubated with shaking at room temperature for 1 hour. Unbound detection antibody is be washed off (7x, PBS 0.05% Tween-20) before TMB substrate is added and the reaction monitored for 1-5 minutes. The reaction is stopped using 1M phosphoric acid and the plate is read at 450 nm. High absorbance is indicative of Rituximab binding and low absorbance is indicative poor binding.

DELFIA assay

The ability of bacteriophage displayed BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP or MP ^m to bind Rituximab is assessed using an ELISA assay. Briefly, Rituximab is coated onto 96- well polystyrene plates at approximately 2 ug/ml overnight at 4 °C. The plates are blocked with DELFIA assay buffer with 1% BSA (v/v) at room temperature for 1 hour. Following washing of the blocking buffer (3x, PBS/ TBS 0.05% Tween-20), bacteriophage displaying BPP, BP ^m P, BPP ^DA , BP ^m P ^DA , MP or MP ^m are added and incubated for 1 hour at room temperature with shaking.

Following washing (5x, TBS 0.05% Tween-20), a biotinylated anti-T7tag antibody (l-in-2000 dilution in TBS) is added and incubated with shaking at room temperature for 1 hour. Following another round of washing (5x, TBS 0.05% Tween- 20), streptavidin-Europium (l-in-10000) is added and incubated with shaking at room temperature for 1 hour. Unbound streptavidin europium is washed off (7x, TBS 0.05% Tween-20) before the enhancement solution is added and the plate is read by fluorescence. Intense fluorescence is indicative of Rituximab binding and low fluorescence is indicate poor binding.

Table 4

Peptide mimotope cassettes

Cassette Description Amino Acid

Sequence

Binding protein-PMCD20- This cassette comprises a binding protein, SEQ ID NO: 48 partner (BPP) PMCD20 and a protein binding partner.

Binding protein- This cassette comprises a binding protein, SEQ ID NO: 49 PMCD20 ^m -partner (BP ^m P) PMCD20 ^m and a protein binding partner.

Binding protein-PMCD20- This cassette comprises a binding protein, SEQ ID NO: 50 partner ^DA (BPP ^DA ) PMCD20 and a protein binding partner

wherein the aspartic acid residue capable

of forming a covalent isopeptide bond is

replaced with a alanine residue.

Binding protein- This cassette comprises a binding protein, SEQ ID NO: 51

PMCD20 ^m -partner ^I PMCD20 ^m and a protein binding partner

(BP ^m P ^DA ) wherein the aspartic acid residue capable

of forming a covalent isopeptide bond is

replaced with an alanine residue.

Modified binding protein- This cassette comprises a modified SEQ ID NO: 52 PMCD20 (MP) binding protein wherein PMCD20 is

inserted into a region of the isopeptide

protein.

Modified binding protein- This cassette comprises a modified SEQ ID NO: 53 PMCD20 ^m (MP ^m ) binding protein wherein PMCD20 ^m is

inserted into a region of the isopeptide

protein.

Example 6: Display of a peptide mimotope

Regulation of mitochondrial outer membrane permeabilization (MOMP) is one of the most critical steps in apoptosis pathways because the release of apoptogenic proteins from the mitochondrial intermembrane space commits the cell to death, either by a caspase-dependent or -independent mechanism. MOMP is regulated by the Bcl-2 family of proteins which comprise both pro- and anti-apoptotic members. Key to regulating the pro-apoptotic pathway are the BH3-domain only protein members including Bim, Bid, and Puma, which activate BAX and Bak. The amino acid sequence of Bim BH3 is set forth in SEQ ID NO: 69. The amino acid sequence of Bid BH3 is set forth in SEQ ID NO: 70. The amino acid sequence of PUMA BH3 is set forth in SEQ ID NO: 71. BAX and Bak proteins are thought to form an oligomeric Mitochondrial Apoptosis-Induced Channel (MAC) in the outer mitochondrial membrane, which in turn results in the release of pro-apoptotic molecules such as cytochrome-c. The importance of the BH3 domains in driving the apoptotic pathways has been recognised with the development of BH3-mimetics targeting some Bcl-2 pro-survival proteins, with molecules showing promise in clinical trials for the treatment of cancers such as leukemia and lymphoma.

Materials and Methods

Constructs

pET28a+ modified binding protein-BimBH3 (MBimBH3), pET28a+ modified binding protein-BidBH3 (MBidBH3), pET28a+ modified binding protein-PumaBH3 (MPumaBH3), pET28a+ binding protein-BimBH3 -protein binding partner (BBimBH3), pET28a+ binding protein-BidBH3- protein binding partner (BBidBH3) and pET28a+ binding protein-BPumaBH3- protein binding partner (BPumaBH3) are synthesized (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 5.

Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector.

Cytochrome-c assay

The ability of MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3 or BPumaBH3 to induce cytochrome c release in the absence of the known direct activators, Bim and Bid is using a cytochrome c release assay as described in Chipuk et al. 2008 and Checco et al. 2015. Briefly, digitonin-permeabilized cell pellets or MEFs are grown in DMEM, harvested and washed with PBS supplemented with 1 mM CaC12 and 1 mM MgC12. Cells are permeabilized by resuspension into a buffer containing 20 mM HEPES, pH 7.4, 250 mM sucrose, 100 mM KC1, 5 mM MgC12, 1 mM EDTA, 1 mM EGTA, and 100 μg/ml digitonin at 3.5 x 107 cells/ml. After incubation on ice for 5 min, 1% BSA is added to remove digitonin, and the cells are pelleted and resuspended in the same volume of buffer without digitonin. This suspension of permeabilized cells is incubated with MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3, BPumaBH3 at 30 °C for 1 h and is spun down at 1000 g for 5 min to separate the supernatant and the pellet. Equivalent amounts of the supernatant and the pellet are loaded onto 15% SDS-PAGE gels for cytochrome c immunoblotting, using an anti- cytochrome c antibody (BD Pharmingen).

Cell viability assay

Cell viability following addition of MBimBH3, MBidBH3, MPumaBH3,

BBimBH3, BBidBH3 or BPumaBH3 is evaluated using the PrestoBlue® Assay (LifeTech). Briefly, cells are seeded into 96 well plates and allowed to settle overnight. MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3 or BPumaBH3 are added to cells at a range of concentration in complete medium. At 24 and 48 h time points a 1/lOth volume of the PrestoBlue® reagent is be added to the media and the cells are incubated for a further 30 min. Fluorescent signal indicating cell metabolic activity are then be measured as per manufacturer's instructions using an EnSpire® multimode plate reader (Perkin Elmer).

Table 5

Peptide mimotope cassettes

Cassette Description Amino Acid

Sequence

Binding protein- BimBH3- This cassette comprises a binding protein, SEQ ID NO: 75 partner (BBimBH3) BimBH3 and a protein binding partner.

Binding protein- BidBH3- This cassette comprises a binding protein, SEQ ID NO: 76 partner (BBidBH3) BidBH3 and a protein binding partner.

Binding protein- This cassette comprises a binding protein, SEQ ID NO: 77 PumaB H3 -partner BPumaBH3 and a protein binding partner.

(BPumaBH3)

Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 72 BimBH3 (MBimBH3) protein wherein BimBH3 is inserted into a

region of the isopeptide protein.

Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 73 BidBH3 (MBidBH3) protein wherein BidBH3 is inserted into a

region of the isopeptide protein.

Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 74 PumaBH3 (MPumaBH3) protein wherein PumaBH3 is inserted into a

region of the isopeptide protein.

Example 7: Display of a peptide aptamer

The signal transducer and activator of transcription, Stat5, is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. A peptide aptamer (PA) has been shown to directly interact with the DNA -binding domain of Stat5. The amino acid sequence of PA is set forth in SEQ ID NO: 54.

Materials and Methods

Constructs

pET28a+ cell penetrating peptide- partner-PA-binding protein (CPPAB), pET28a+ cell penetrating peptide- partner ^DA -PA-binding protein (CP ^DA PAB), pET28a+ partner -PA-binding protein -cell penetrating peptide (PPABC), pET28a+ partner ^DA - PA-binding protein -cell penetrating peptide (P ^DA PABC), pET28a+ partner-PA-binding protein (PPAB), pET28a+ partner ^DA -PA-binding protein (P ^DA PAB), pET28a+ partner - PA-binding protein (PPAB), pET28a+ partner ^DA -PA-binding protein (P ^DA PAB), pET28a+ modified binding protein-PA (MPA) and pET28a+ cell penetrating peptide- modified binding protein-PA (CMPA) are synthesized (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). The cassettes include a hexahistidine tag to aid purification. A description of the cassettes (including the amino acid sequence) is provided in Table 6.

Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector. Cell transfection

Transient transfection is performed as previously described by Weber et al.

2013.

Dual-luciferase reporter assay

A dual-luciferase reporter assay is performed as previously described by Weber et al. 2013. Table 6

Peptide aptamer cassettes

Cassette Description Amino Acid

Sequence pET28a+ cell penetrating This cassette comprises a cell penetrating SEQ ID NO: 84 peptide- partner-PA- peptide, a protein binding partner, a peptide

binding protein (CPPAB) aptamer and a binding protein.

pET28a+ cell penetrating This cassette comprises a cell penetrating SEQ ID NO: 85 peptide- partnerDA-PA- peptide, a protein binding partner wherein

binding protein the aspartic acid residue capable of forming

(CP ^DA PAB) a covalent isopeptide bond is replaced with

an alanine residue, a peptide aptamer and a

binding protein.

pET28a+ partner -PA- This cassette comprises a protein binding SEQ ID NO: 86 binding protein-cell partner, a peptide aptamer, a binding

penetrating peptide protein and a cell penetrating peptide.

(PPABC)

pET28a+ partner ^DA -PA- This cassette comprises a protein binding SEQ ID NO: 87 binding protein -cell partner wherein the aspartic acid residue

penetrating peptide capable of forming a covalent isopeptide

(P ^DA PABC) bond is replaced with an alanine residue, a

peptide aptamer, a binding protein and a

cell penetrating peptide.

pET28a+ partner-PA- This cassette comprises a protein binding SEQ ID NO: 88 binding protein (PPAB) partner, a peptide aptamer and a binding

protein.

pET28a+ partner ^DA -PA- This cassette comprises a protein binding SEQ ID NO: 89 binding protein (P ^DA PAB) partner wherein the aspartic acid residue

capable of forming a covalent isopeptide

bond is replaced with an alanine residue, a

peptide aptamer and a binding protein.

pET28a+ modified This cassette comprises a modified binding SEQ ID NO: 56 binding protein-PA protein wherein a protein aptamer is

(MPA) inserted into a region of the isopeptide

protein. Cassette Description Amino Acid

Sequence pET28a+ cell penetrating This cassette comprises a cell penetrating SEQ ID NO: 57 peptide-modified binding peptide and a modified binding protein

protein-PA (CMP A) wherein a protein aptamer is inserted into a

region of the isopeptide protein.

Example 8: Display of a cyclic peptide

The human salivary peptide histatin 1 (Hstl), which accelerates re-epithelization in simple and complex wound models, is an example of a linear peptide of which the molar activity increases a 1000-fold on cyclization as described, for example, in Bolscher et al. 2011. The amino acid sequence of Hstl is set forth in SEQ ID NO: 78. Histatins, rather than EGF, are the major wound-closing factors in human saliva. Characterization of histatins indicates that their activation has several features in common with "classic" growth factors. These include stereo specific and active uptake by the cell and the requirement of a specific intracellular signaling pathway [extracellular signal-regulated kinases 1/2 (ERK1/2)].

Materials and methods

Constructs

pET28a+ binding protein-Hstl -protein binding partner (BHP), pET28a+ binding protein-Hstl (BH) and pET28a+ modified binding protein-Hstl (MH) are synthesized are synthesized (DNA 2.0, Menlo Park, CA, USA). The synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 7.

Protein expression and purification

BHP, BH and MH are expressed and purified using the method described in Example 1.

Peptide synthesis

Synthetic Hst-1 (SHstl) is synthesised using solid phase peptide synthesis techniques described in Example 1. In vitro wound closure assays

The biological activity of BHP, BH and MH is evaluated in an artificial wound model as previously described by Bolscher et al. 2011. Briefly, in vitro wound closure experiments are performed in 12, 48 or 96-well culture plates with human buccal epithelial cells (HO-l-N-1). A sterile tip is used to make a scratch in a confluent layer of cells that are serum deprived in DMEM/F12 culture medium (Invitrogen, Carlsbad, CA, USA) for 6 h. The width of the scratch is measured at the beginning and after 18 h of culture on microscopic images. Titred BHP, BH and MH (1 nM to 20 uM) are added to the wells to determine whether the cyclized Histatin is more potent than the linear displayed peptide. Relative closure will be calculated by dividing the closure in the presence of peptide by that in the absence of peptide.

The biological activity of BHP, BH and MH is also evaluated using wound- closure experiments as previously described by Oudhoff et al. 2008. Briefly TR146 cells are grown in 12-, 24-, or 48-well plates until confluence, and serum deprived for 24 h in keratinocyte serum-free medium (SFM; Invitrogen). In each well a scratch is made using a sterile tip, and cellular debris is removed by washing with SFM. The width of the scratch is determined microscopically immediately after creation and 16 h later. The effects of the following conditions on wound closure is analyzed:

• SHstl, dissolved in SFM, at final concentrations of 1 nM to 10 uM

· BHP dissolved in SFM, at final concentration of 1 nM to 10 uM

• BH, at final concentrations of 1 nM to 10 uM.

• MH, dissolved in SFM, at final concentration of 1 nM to 10 uM

• SFM will be used as a negative control. Relative closure is calculated as (X0_X16h)/(C0_C16h), where X0 _ width of the scratch at time 0, X16h _ width of the scratch after 16 h exposure to a condition, CO _ width of the scratch at time 0, and C16h _ width of the scratch after 16 h exposure to the control (saliva buffer or SFM). Table 7

Cyclic peptide cassettes

Cassette Description Amino Acid

Sequence pET28a+ binding protein This cassette comprises a binding protein, SEQ ID NO: 80 -Histl- partner (BHP) Histl and a protein binding partner.

pET28a+ binding protein- This cassette comprises a binding protein, SEQ ID NO: 81 Histl (BHP) Histl.

pET28a+ modified This cassette comprises a modified binding SEQ ID NO: 79 binding protein-Histl protein wherein Histl is inserted into a

(MH) region of the isopeptide protein.

Example 9: Identification of peptides that interact with a molecule

Material and Methods

Bacteriophage display library

A bacteriophage display library is generated and labelled using the method described in Example 1.

Biopanning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.

Example 10: Identification of peptides that interact with Epidermal growth factor receptor (EGFR) Material and Methods

Bacteriophage display library

A bacteriophage display library is generated and labelled using the method described in Example 1. Cells

In one example, a Chinese hamster ovarian cell line, CHO-Kl, is used for negative selection. CHO-Kl cells do not express EGFR. A CHO-Kl cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.

In one example, a human embryonic kidney (HEK)293 cell line, is used for negative selection. HEK293 cells do not express EGFR. A HEK293 cell line expressing EGFR EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.

Biopanning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007. Example 11: Identification of peptides that interact with the Ephrin receptor EphA2

Material and Methods

Bacteriophage display library

A bacteriophage display library is generated and labelled using the method described in Example 1.

Cells

In one example, a Chinese hamster ovarian cell line, CHO-Kl, is used for negative selection. A CHO-Kl cell line over-expressing the Ephrin receptor EphA2 is used for positive selection.

Bio-panning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.

Example 12: Identification of peptides that interact with the CXCR4 receptor

Bacteriophage display library

A bacteriophage display library is generated and labelled using the method described in Example 1. Cells

In one example, a Chinese hamster ovarian cell line, CHO-K1, is used for negative selection. CHO-K1 cells do not express the CXCR4 receptor. A CHO-K1 cell line over-expressing the CXCR4 receptor is used for positive selection.

Bio-panning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.

Example 13: Identification of peptides capable of translocating a membrane of a cell

Material and Methods

Bacteriophage display library

A bacteriophage display library is generated using the method described in Example 1.

Bio-panning

In brief, phage display libraries are incubated with HEK-293, CHO-K1, NIH- 3T3, HeLa or COS-7 cells. After treatment to remove surface-bound phage, cells are harvested, either by trypsinization or cell scraping, and then lyzed to recover internalized phage. Between 1 to 5 iterative rounds of biopanning were performed for each screen.

Peptides are identified by recovering the bacteriophage displaying the modified binding proteins and determining the nucleic acid sequence of the modified binding proteins. The deduced amino acid sequences of the peptides within the modified binding protein are then analyzed by:

(i) pairwise alignment using the CD Hit clustering program;

(ii) characterization of the peptides for amphipathicity, hydrophobicity, charge, size, and amino acid composition e.g., presence of arginine and lysine residues;

(iii) characterization of predicted secondary structures; and

(iv) database query to determine novelty of the peptides.

Bioinformatics employed PSIPRED algorithm. Database queries are performed using a database of known CPPs available at the database "CellPPD: Designing of Cell Penetrating Peptides", which provides in silico prediction of cell penetration efficiency based on a dataset of 708 experimentally- validated CPPs. In particular, CellPPD permits prediction of peptides having CPP-like properties in each pool of isolated or identified peptides based on their sequences, including the identification of CPP-like motifs in peptides.

Example 14: Identification of receptor binding domain that interact with Epidermal growth factor receptor (EGFR) using a complex comprising a modified binding protein and a binding protein partner linked to a cell-penetrating peptide

Materials and Methods:

Bacteriophage display library

A bacteriophage display library is generated using the method described in

Example 1 and labelled with either SEQ ID 92 or SEQ ID 93.

Cells

In one example, a Chinese hamster ovarian cell line, CHO-K1, is used for negative selection. CHO-K1 cells do not express EGFR. A CHO-K1 cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.

In one example, a human embryonic kidney (HEK)293 cell line, is used for negative selection. HEK293 cells do not express EGFR. A HEK293 cell line expressing EGFR EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.

Biopanning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.

Example 15: Identification of a peptide capable of translocating a membrane of a cell using a complex comprising a modified binding protein linked to an EGRF binding Affibody

Material and Methods

Generation of EGFR Affibody modified binding protein (RMP) cassettes for use in bacteriophage display

For the display of a nucleic acid fragment library in any of the designated regions of the modified binding protein, several MP library cassettes are generated using pET28+ as a template, where the EGFR Affibody receptor-binding-domain is either N- or C-terminal to the modified binding protein, named RMP library and MPR library cassettes respectively. Briefly, the RMP library and MPR library cassettes contain an EcoRI restriction site in the designated loop region that includes a frameshift base that shifts the reading frame to ablate correct translation of modified binding protein 3' to the EcoRI cloning site when no DNA is inserted. The nucleic acid sequences of the RMP library and MPR library cassettes are set forth in SEQ ID NO: 94 and 95 respectively.

To create a T7 display vector, the RMP library and MPR library cassettes are cloned into T7-Prescission-Avi cut with EcoRI/ Sail. The resulting vectors are designated, T7-RMP-Entry and T7-MPR-Entry.

Bacteriophage display library

A bacteriophage display library is generated and labelled using the method described in Example 1.

Cells

In one example, a Chinese hamster ovarian cell line, CHO-K1, is used for negative selection. CHO-K1 cells do not express EGFR. A CHO-K1 cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.

In one example, a human embryonic kidney (HEK)293 cell line, is used for negative selection. HEK293 cells do not express EGFR. A HEK293 cell line expressing EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.

Biopanning

Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007. In brief, phage display libraries are incubated with HEK-293-EGFR or CHO- Kl-EGFR cells. After treatment to remove surface-bound phage, cells are harvested, either by trypsinization or cell scraping, and then lyzed to recover internalized phage. Between 1 to 5 iterative rounds of biopanning were performed for each screen.

Peptides are identified by recovering the bacteriophage displaying the modified binding proteins and determining the nucleic acid sequence of the modified binding proteins. The deduced amino acid sequences of the peptides within the modified binding protein are then analyzed by:

(i) pairwise alignment using the CD Hit clustering program; (ii) characterization of the peptides for amphipathicity, hydrophobicity, charge, size, and amino acid composition e.g., presence of arginine and lysine residues;

(iii) characterization of predicted secondary structures; and

(iv) database query to determine novelty of the peptides.

Bioinformatics employed PSIPRED algorithm. Database queries are performed using a database of known CPPs available at the database "CellPPD: Designing of Cell Penetrating Peptides", which provides in silico prediction of cell penetration efficiency based on a dataset of 708 experimentally- validated CPPs. In particular, CellPPD permits prediction of peptides having CPP-like properties in each pool of isolated or identified peptides based on their sequences, including the identification of CPP-like motifs in peptides.

Example 16: Display of a protein albumin binding domain (ABD)

Human serum albumin (HSA) is the most abundant protein in blood, with a circulatory concentration of ~600μΜ. It is responsible for the transport of various chemical entities including fatty acids/ lipids and hormones through the vasculature, and further plays a role in pH buffering of blood. HSA is a large protein of about 66.5kDa and is stable with a serum half-life of 19 days. Non-covalent linking to HSA has been shown to be an effective way to improve the pharmacokinetic properties of short-lived peptides. This interaction has the effect of making short peptides appear much larger (>60kDa) which reduces excretion via glomerular filtration by the kidneys, and thus increasing the serum half-life of the biomolecule.

Numerous Gram-positive bacterial species, including human pathogens, express surface proteins that interact with host proteins like human serum albumin (HSA) and IgG with high specificity and affinity. Protein G binds to both IgG and HSA, and the regions that do so are separately located on the molecule. The region responsible for binding to HSA is referred to as the GA module and is highly conserved.

Jacobs et al. 2015, designed an albumin-binding domain based on the consensus sequence of 20 homologs of the GA module, and named it ABDcon. Jacobs et al. 2015, also mutated key residues in the ADBcon consensus sequence to tune the binding to Human Serum Albumin (HSA), Murine Serum Albumin (MSA) and Rat Serum Albumin (RSA).

N-terminal extension of the consensus sequence with TIDEWL (SEQ ID NO: 130) improves thermal stability through N-terminal helix extension and hydrogen- bond formation (Jacobs et al 2015, Lejon et al 2004 and Johansson et al 2002). The 1TF0 crystal structure also indicates that C-terminal extension with two residues (HA) extends the C-terminal helix of the ABD and appears to stabilise the interaction of HSA and the ABD.

Dennis et al. 2002, reported a family of multispecies serum albumin binding specific peptides with a core sequence of DICLPRWGCLW (SEQ ID NO: 125) that was identified by phage display using a range of cysteine constrained random peptide libraries. Two key peptides, SA20 and SA21 have been further validated by Angelini et al 2012 and Langerheim et al 2009.

Grafting the albumin binding domains into one of the loops (e.g., the L2 loop) of the modified binding protein allows for the incorporation of a half-life extension functionality that allows the protein binding partner modality to be retained in trans for other applications such as cell-penetrating peptide, fluorophore, small molecule addition etc. The albumin binding domains selected for display in the L2 Loop of the modified binding protein were: ABDcon (SEQ ID NO:98), ABDcon5 (SEQ ID NO:99), ABDcon9 (SEQ ID NO: 100) and SA20 (SEQ ID NO: 104).

The albumin binding ability of the albumin binding domain displaying modified binding proteins was evaluated by an HSA binding affinity assay. To determine the impact on the albumin binding affinity of the above sequences being displayed in the loop of the modified binding protein, linear controls coupled to the protein binding partner were synthesized as peptides and ligated to the binding protein. These complexes were then evaluated in the HSA binding affinity assay in parallel to the modified binding proteins

Peptide Constructs:

Albumin binding domain sequences, ABDcon5, ABDcon9 and SA20 coupled to the protein binding partner sequence on the N-terminal, respectively, were synthesized as peptides (Mimotopes, Clayton, Victoria, Australia). A description of the peptides and a list of their corresponding SEQ ID Nos is provided in Table 8.

Table 8 Description of ABD peptides

Protein Constructs:

pET28a+ modified binding protein- ABDcon (MABDcon), pET28a+ modified binding protein-ABDcon5 (MABDcon5), pET28a+ modified binding protein- ABDcon9 (MABDcon9), pET28a+ modified binding protein-SA20 (MSA20) and pET28a+ binding protein (B) were codon optimized for E.coli expression and synthesized (DNA 2.0, Menlo Park, CA, USA). The synthesized cassettes were cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). The cassettes include a hexahistidine tag and prescission protease cleavage site to aid purification. A description of the peptides and a list of their corresponding SEQ ID Nos is provided in Table 9.

Table 9 Description of pET28a+ modified binding proteins containing ABDs

Name Description Amino Acid

Sequence

Modified binding protein- This cassette comprises a modified SEQ ID NO: 105 pep tide ABDcon binding protein wherein ABDcon is

(MABDcon) inserted into a region of the

isopeptide protein.

Modified binding protein- This cassette comprises a modified SEQ ID No: 106 pep tide ABDcon5 binding protein wherein ABDcon5 is

(MABDcon5) inserted into a region of the

isopeptide protein. Name Description Amino Acid

Sequence

Modified binding protein- This cassette comprises a modified SEQ ID NO: 107 pep tide ABDcon9 binding protein wherein ABDcon9 is

(MABDcon9) inserted into a region of the

isopeptide protein.

Modified binding protein- This cassette comprises a modified SEQ ID NO: 108 peptide SA20 (MSA20) binding protein wherein ABDcon9 is

inserted into a region of the

isopeptide protein.

Binding protein This cassette comprises a binding SEQ ID NO: 14 protein

Protein expression and purification

MABDcon, MABDcon5, MABDcon9 and MSA20 were expressed and purified using the method described in Example 1.

Modified binding protein, binding protein and protein binding partner conjugations

To test the ability of MABDcon, MABCon5, MABDcon9 and MSA20 to interact with a protein binding partner, was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio.

In one example, MABDcon was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.

In one example MABDcon5 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.

In one example MABDcon9 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.

In one example MSA20 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.

The reaction mixture was analysed by SDS PAGE under low light conditions. The gels are also then read under a FITC channel. Gels were also stained with Coomassie blue stain. The efficiency of the interactions between MABDcon and FITC- Partner, MABDcon9 and FITC-Partner, MABDcon9 and FITC-Partner, and MSA20 and FITC-Partner was determined by comparing the molecular mass of MABDcon, MABDcon5, MABDcon9 or MSA20 alone or FITC-Partner alone with the molecular mass of a sample of the reaction mixture comprising MABDcon and FITC-Partner, MABDcon9 and FITC-Partner, MABDcon9 and FITC-Partner, and MSA20 and FITC- Partner, respectively. In addition, densitometry was used to evaluate conjugation reaction efficiency.

To generate unconstrained albumin binding domains, partner-ABDcon5, partner-ABDcon9 and partner-SA20 synthetic peptides were conjugated to binding protein by mixing at a peptide: protein stoichiometric ratio of 1.1: 1, to generate B- partner-ABDcon5, B-partner-ABDcon9 and B-partner-SA20 conjugates respectively. Reactions were incubated at 37°C for 3 h or at 4 °C overnight. The reaction was then stopped. Human Serum Albumin binding assay

The ability of MABDcon, MABDcon5, MABDcon9, MSA20, B-partner- ABDcon5 conjugate, B -partner- ABDcon9 conjugate and B-partner-SA20 conjugate, to interact with HSA was evaluated by biolayer interferometry using the Octet®RED platform (Pall ForteBio LLC, Menlo Park, CA, USA).

In brief, biolayer interferometry was used to measure the capture of biotinylated

HSA (bio-HSA) on streptavidin biosensors (step 1). The biosensors were then incubated with solutions containing various concentrations of the albumin binding domain containing modified binding proteins, and binding protein - protein binding partner conjugates, respectively, to measure the rates of association of the proteins to bio-HSA (Step 2). The biosensors were then moved from the protein solutions into assay buffer to measure the rates of dissociation (Step 3). Together, the rates of association in step 2 and rates of dissociation in step 3 were used to determine the binding affinities of the various proteins, respectively, according the manufacturer's instructions (Pall ForteBio).

Binding affinity was measured in molar terms, with increased affinity indicated by decreasing concentration.

Results:

Modified binding protein, binding protein and protein binding partner conjugations MABDcon formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -24 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon-FITC- partner conjugate, in comparison to the unconjugated MABDcon molecular weight of ~20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).

MABDcon5 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -24 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon5- FITC-partner conjugate, in comparison to the unconjugated MABDcon5 molecular weight of ~20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon5 is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).

MABDcon9 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at ~24kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon9- FITC-partner conjugate, in comparison to the unconjugated MABDcon9 molecular weight of ~20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon9 is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).

MSA20 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -20 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MSA20-FITC- partner conjugate, in comparison to the unconjugated MSA20 molecular weight of ~16kDa. This confirms that the modified binding protein was able to fold correctly when SA20 is displayed in the modified site, and retain its ability to conjugate to FITC- partner (Figure 13).

MABDcon when incubated with various concentrations of bio-HSA was determined to have a binding affinity of 9.1nM by biolayer interferometry, indicating that the ABDcon sequence retain its high affinity to HSA when displayed in the modified binding protein (Figure 14).

MABDcon5 when incubated with various concentrations of bio-HSA was determined to have a binding affinity of 58.7nM by biolayer interferometry (Figure 14). B-partner-ABDcon5 conjugate when incubated with various concentration of bio-HSA was determined to have a binding affinity of 20nM by biolayer interferometry

(Figure 15). These data indicate that the ABDcon5 sequence has an approximately three-fold weaker affinity to HSA when displayed in the modified binding protein, but is still able to bind with a 58.7nM affinity (Figure 14).

MABDcon9 when incubated with various concentrations of bio-HS A was determined to have a binding affinity of 75.9 nM by biolayer interferometry

(Figure 14). B-partner-ABDcon9 conjugate when incubated with various concentration of bio-HS A was determined to have a binding affinity of 60 nM by biolayer

interferometry (Figure 15). These data indicate that the ABDcon9 sequence has an approximately 1.3-fold weaker affinity to HSA when displayed in the modified binding protein, but is still able to bind with a 75.9 nM affinity.

MSA20 when incubated with various concentrations of bio-HSA was determined to have a binding affinity of 79.7 nM by biolayer interferometry

(Figure 14), indicating that the SA20 sequence is able to bind to HSA with high affinity when displayed in the modified binding protein.

Binding protein control, B, does not bind to bio-HSA as determined by biolayer interferometry (Figure 15).

Example 17: Bacteriophage display of a Phylomer peptide library constrained by interaction between a binding protein and a binding protein partner

Compared to linear peptides, cyclic peptides have been shown to be superior protein interaction partners due to i) more biologically relevant conformations, ii) increased structural rigidity and iii) higher stability due to reduced proteolytic attack. By displaying a Phylomer peptide library downstream of a binding protein and upstream of a binding protein partner, a subset of the Phylomer peptides will be presented as cyclic peptides when the binding protein partner forms an intramolecular, covalent bond with the preceding binding protein. A Phylomer library containing such cyclic peptides is expected to provide superior interaction partners for targets, which require a structured interaction partner. One configuration of such a library is schematically illustrated in Figure 16. Materials and Methods

Generation of binding protein-binding protein partner library cassette (BP_BPP_lib) or use in bacteriophage display

For the display of a nucleic acid fragment library between a binding protein and a binding protein partner, a BP_BPP_lib cassette was generated in pET28a+. The

BP_BPP_lib cassette comprises an EcoRI restriction site between nucleic acids encoding the binding protein and nucleic acids encoding the binding protein partner. The EcoRI site is followed by two extra bases that shift the reading frame to ablate correct translation of the binding protein partner when no DNA is inserted into the EcoRI restriction site. The nucleic acid sequence and translated amino acid sequence of the BP_BPP_lib cassette are set forth in SEQ ID NOs: 109 and 110, respectively.

Generation of bacteriophage T7 library vector

An Mfel/Xhol BP_BPP_lib DNA fragment was isolated and cloned into EcoRI/XhoI sites of bacteriophage T7-Prescission-Avi DNA as described in Example 1.

Generation of Phylomer peptide library constrained by interaction between a binding protein and a binding protein partner

The T7-Prescission-Avi-BP_BPP_lib vector was used to generate the T7 Phylomer library T16, in which the library inserts were flanked at the N-terminus by binding protein and by a binding protein partner at the C-terminus. The library was generated as described in Example 1.

T16 library inserts were generated from the fully sequenced genomes of eight archaebacterial genomes, namely Aeropyrum pernix, Archaeoglobus fulgidus, Haloarcula marismortui, Halobacterium salinarum, Methanocaldococcus jannaschii, Pyrococcus horikoshii, Sulfolobus solfataricus and Thermoplasma volcanium.

Due to the random fragment generation process, the library inserts had varying insert lengths. Only inserts of the length 3n+l allowed in-frame translation of the C- terminal binding protein partner. The random fragment generation process also produced inserts with stop codons leading to non-readthrough into the DNA of the C- terminal binding protein partner.

Phage binding to M280 magnetic beads coated with synthetic binding protein partner

500 μΐ of the naive T16 library were amplified in 25 ml of the host strain BLT5615/pSUMO-avi3, which greatly reduced biotinylation of the avi-tag displayed on the T7 phage. 0.5 ml of the amplified, PEG-precipitated T16 library were further cleared of pre-biotinylated T7-phage by passing over a column containing 300 ul of Streptavidin Sepharose High Performance resin (GE Healthcare, cat#17-5113-0). The phage titer after clearing was 6.3 x 10 ¹¹ pfu/ml.

100 μΐ of M-280 paramagnetic Streptavidin beads (Invitrogen, cat#11206D) were washed with 1 ml of PBS-0.05%-Tween-20 (PBS-T) and buffer aspirated. 1 ml of 1% BSA-PBS-0.05% Tween were added to the beads and blocked overnight at 4°C with rotation. The blocking buffer was aspirated, the beads were washed once in 1 ml of PBS and resuspended in 90 ul of PBS.

10 μΐ of a 1 mM solution of biotinylated binding protein partner (SEQ ID NO 65) was added to the blocked beads and incubated at 1 h with rotation to allow for binding of the binding protein partner to the Streptavidin groups on the surface of the beads. After 1 h, 100 μΐ of a 0.5 mM solution of free D-biotin (Sigma, cat #B4501) was added to saturate any remaining free Streptavidin binding sites. After an additional 30 min of rotation, the beads were washed twice with 1 ml of PBS/0.05% Tween-20. The washed beads were finally resuspended in 100 μΐ of PBS/0.05% Tween-20.

50 μΐ of binding protein partner-coated and blocked beads were incubated with

1 x 10 ⁹ pfu of cleared T7 library in PBS/0.05% Tween-20 in a volume of 300 μΐ. Phage and beads were incubated for 3 h under rotation. The beads were washed three times with 1 ml of PBS/0.05% Tween-20 and finally resuspended in 44 ul of PBS.

To release captured phage from the beads, 5 ul of 10X HRV3C Protease buffer and 1 ul of HRV3C protease (EMD Millipore Cat #71493) were added and the reaction was incubated at room temperature for 2 hours under rotation. The supernatant containing the released phage was aspirated and the phage titer determined.

The amplified starting phage library was named T16_Amp, the bead-captured phage fraction was named T16_Acapt and the non-captured phage fraction T16_Acycl.

Library characterisation by Next Generation sequencing

The starting phage library, T16_Amp and the fractions from the bead binding experiment T16_Acapt and T16-Acycl were analysed by 250 bp Illumina paired-end MiSeq. The samples were prepared for sequencing by the 2-step PCR described in the Illumina manual "16S Metagenomic Sequencing Library Preparation Guide". Due to low phage numbers, the complete volume of T16_Acapt was amplified in 2.5 ml of BLT5615 host culture before sequencing.

Amplicon PCR

1 μΐ of each phage fraction was used as a template for Amplicon PCR as described in the Illumina manual. The reactions were carried out with equimolar mixtures of four staggered forward and four staggered reverse primers as shown in SEQ ID NOs: 116-119.

Oligonucleotides were synthesised by Sigma as desalted PCR primers. The PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent

Cat#600675). Thermocycling conditions were: 5 min at 95°C + 20 cycles [20 sec at

95°C, 20 sec at 60°C, 1 min at 72°C] + 3 min at 72°C. The reactions were cleaned up by Agencourt AMPure XP PCR Purification beads (Beckman Coulter Cat#A63880) as described by the manufacturer.

Index PCR

1 μΐ of each purified Amplicon PCR was used as a template for an Index PCR, carried out as described in the Illumina manual with index primers from the

recommended Nextera XT Index Kit (Illumina Cat#FC-131-1001). The PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent Cat#600675). Thermocycling conditions were: 2 min at 95°C + 8 cycles [20 sec at 95°C, 20 sec at 58°C, 1 min at 72°C] + 3 min at 72°C. The reactions were cleaned up by Agencourt AMPure XP PCR Purification beads (Beckman Coulter Cat#A63880) as described by the manufacturer.

Samples were submitted to the Australian Genome Research Facility for sequencing by paired-end 250 bp Illumina MiSeq. Samples were loaded at 600 K/mm cluster density with 15% PhiX. The quality of the raw data was confirmed by FastQC. The paired-end sequencing reads were converted into Phylomer DNA and Phylomer peptide sequences by the in-house software PhySeq4NGS.

Results:

Characterisation of T16_Amp

The complexity of the Phylomer peptide library T16 was determined as 2.6 x 10 by phage titering before library amplification; the titer of the amplified library was 5.2 x 10 ¹¹ pfu/ml.

Several library parameters were determined by NGS. As a form of quality control, NGS was used to assess the genome coverage in the library.

Table 10 shows the genomes covered in T16_Amp. Fold-difference: Observed percentage divided by expected percentage. ^Expected representation is achieved if fold-difference is between 0.67 and 1.5. Over-representation if fold-difference >1.5; under-representation if fold-difference <0.67.

Table 10 shows that T16_Amp contained all eight archaebacterial genomes that were inserted into the library. For library cloning, the genomic fragments were pooled according to genome sizes to achieve equal coverage of genomic loci resulting in different expected percentage values for each genome. The observed proportion of genomic fragments from Methanocaldococcus jannaschii in the T16 library was 1.8- fold higher than expected, while the remaining seven genomes were in the expected range with four genomes slightly underrepresented. Overall, the

over/underrepresentation of genomes observed in T16 was in an acceptable range.

NGS also allowed the assessment of the library diversity. All peptide sequences of 6 aa and longer were clustered at 80% homology to work out the number of unique peptide sequences (Table 11); the copy numbers of all unique sequences are also shown in Table 11. The diversity is reflected in the "diversity number", which is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. The fact that 93% of all unique peptides occurred only once and a diversity number of 0.89 for T16_Amp indicated a very good diversity.

TABLE 11 Summary of Sequence Diversity in T16 Library

Table 11 provides a summary of numbers of peptide sequences identified by NGS. The diversity number is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. Copy numbers for unique peptide sequence indicate the percentage of unique sequence that were identified once, twice etc.

Further T16 library parameters are summarised in Table 12. All of the general peptide parameters were in the normal range for Phylomer libraries. The mean DNA insert length was determined as 222 bp, which translates into a peptide length of 74 aa. The mean observed peptide length, however, was only 20.5 aa indicating the presence of non-readthrough inserts caused by stop-codons in the randomly- amplified g fragments.

Table 12 provides a summary of DNA insert lengths, display peptide lengths and percentage of annotated open reading frames (ORF).

The length of each library insert was separately assessed to determine whether the translated peptide was in-frame (IF) or out-of-frame (OOF) with the C-terminal binding protein partner. In the case of stop codons in the reading frame, inserts were classified as non-readthrough (nonRT).

Almost 90% of the inserts did not read through into the DNA of the binding protein partner and were therefore not cyclised. A further 5.7% did read through into the DNA of the binding protein partner but the translation was out-of-frame resulting in an unrelated peptide fusion that could not cyclise. 4.4% of the Phylomers were classified as readthrough in-frame (RTIF), translating the protein binding partner downstream of the Phylomer and were therefore potentially cyclised by virtue of the binding protein partner covalently binding to the binding protein. It is however unlikely that all of the RTIF phage clones displayed cyclised Phylomer peptides because very long peptides or peptides with secondary structure could possibly hinder the interaction between binding protein and binding protein partner. The bead binding experiment shown below was devised to assess the proportion of actual cyclisation in T16_Amp. Bead binding experiments

A sample of amplified T16_Amp library was subjected to a binding experiment with immobilised synthetic binding protein partner in order to gain insight into the actual proportion of cyclised Phylomers in the library. Cyclisation via binding and binding protein partner results in an occupied binding pocket of the binding protein so that the phage clones cannot bind to immobilised binding protein partner, while phage clones with non-cyclised Phylomers should be able to bind to immobilised binding protein partner(see Figure 16). The experiment was carried out with 1 x 10 pfu of phage library and a high amount of binding protein partner immobilised on magnetic beads that should have theoretically allowed the capture of lOOfold more uncyclised phage.

Phage titering of the capture fraction and the supernatant after the capture experiment (Table 13) showed that the capture was not quantitative, because >95% of the library clones were not cyclised (being nonRT or RTOOF) and should therefore have bound to the immobilised binding protein partner (Represented by T16_Acapt).

The observed capture rate of 1.8% was far below the expected rate of 95% and indicated inefficient phage capture. Inefficient capture by magnetic beads had been shown earlier and was probably due to steric hindrance.

The problem of inefficient bead capture was compounded by incomplete protease release of captured phage from the beads, which is shown by the fact that phage numbers in T16_Acycl and T16_Acapt did not add up to 100%. In spite of this shortcoming, all the phage fractions were analysed by NGS to assess whether there were qualititative differences between T16_Acycl and and T16_Acapt.

Table 13 Summary of T16 Library Bead Capture Enrichment

Table 13 shows phage titer results of bead binding experiment. Phage numbers in T16_Acycl (supernatant after bead capture) and T16_Acapt (bead-captured fraction) should have added up to 100%. The low level of subtraction indicates inefficient bead capture while the apparent loss of phage indicates inefficient release of captured phage from the magnetic beads.

Comparison of the NGS-derived parameters of T16_Amp, T16_Acycl and T16_Acapt revealed that the proportion of RTIF clones was one of the main differences between the two fractions obtained by the bead binding experiment.

Compared to the starting library, the proportion of RTIF phage clones in T16_Acapt was reduced by 3.1% (from 4.4 to 1.3%), while the proportion of RTIF phage clones in T16_Acycl was equal or marginally higher than in the starting library. This suggests that the bead-immobilised binding protein partner had successfully captured non-cyclised phage clones and that approximately 3% of the overal T16_Amp were cyclised by the interaction between binding protein and binding protein partner and could therefore not be captured on the beads.

Analysis of the peptide lengths of RTIF phage clones showed that the peptide length distribution in the starting library was very similar to the length distribution in the bead experiment fraction indicating that cyclisation did not favour a particular peptide length.

Example 18: Bacteriophage display of a Phylomer peptide library constrained by display in the loop of a modified binding protein

Compared to linear peptides, cyclic peptides have been shown to be superior protein interaction partners due to i) more biologically relevant conformations, ii) increased structural rigidity and iii) higher stability due to reduced proteolytic attack.

By displaying a Phylomer peptide library in a loop of a modified binding protein, the peptides will be presented as cyclic peptides while the binding activity of the binding protein should be retained. A functional binding protein allows additional conjugation of functional groups via binding protein partner, e.g. receptor-binding domains or reporter enzymes. A Phylomer library containing such cyclic peptides is expected to provide superior interaction partners for targets, which require a structured interaction partner. Such a library configuration is schematically illustrated in Figure 17.

Materials and Methods

Generation of modified binding protein-loop display library cassette (M_Loop2_lib) or use in bacteriophage display

For the display of a nucleic acid fragment library in loop 2 of the modified binding protein, a M_Loop2_lib cassette was generated in pET28a+. The M_Loop2_lib cassette comprises an Mfel site followed by the codons for the first 28 aa (strands 1-2) of binding protein. This is followed by an insertion encoding a 5 aa GSGAS-Linker (SEQ ID NO: 123), an EcoRI restriction site and a second, 5 aa QGTGS-Linker (SEQ ID NO: 124). The insertion is followed by a DNA fragment encoding strands 3-8 (59 aa) of binding protein followed by 2 amino acids encoded by a Sail restriction site. Two extra bases have been inserted after the EcoRI site to shift the reading frame to ablate correct translation of the binding protein partner when no DNA is inserted into the EcoRI restriction site. The nucleic acid sequence and amino acid translation of the M_Loop2_lib cassette are set forth in SEQ ID NOs: 120 and 121, respectively. Generation of bacteriophage T7 library vector

The Mfel/Xhol M_Loop2_lib DNA fragment was isolated and cloned into EcoRI/XhoI sites of bacteriophage T7-Prescission-Avi DNA as described in Example 1.

Generation of Phylomer peptide library constrained by modified binding protein- loop display

The T7-Prescission-Avi-M_Loop2_lib vector was used to generate the T7 Phylomer library T18, in which the library inserts were displayed on loop 2 of the modified binding protein. The library was generated as described in Example 1.

T18 library inserts were generated from the fully sequenced genomes of eight archaebacterial genomes, namely Aeropyrum pernix, Archaeoglobus fulgidus, Haloarcula marismortui, Halobacterium salinarum, Methanocaldococcus jannaschii, Pyrococcus horikoshii, Sulfolobus solfataricus and Thermoplasma volcanium.

Due to the random fragment generation process, the library inserts had varying insert lengths. Only inserts of the length 3n+l allowed in-frame translation of the second part of the binding protein. The random fragment generation process also produced inserts with stop codons leading to non-readthrough into the DNA of the second part of the binding protein.

Phage binding to M280 magnetic beads coated with synthetic binding protein partner

500 μΐ of the naive T18 library were amplified in 25 ml of "decoy" host strain

BLT5615/pSUMO-avi3, which greatly reduced biotinylation of the avi-tag displayed on the T7 phage. 0.5 ml of the amplified, PEG-precipitated T18 library were further cleared of pre-biotinylated T7-phage by passing over a column containing 300 μΐ of

Streptavidin Sepharose High Performance resin (GE Healthcare, cat#17-5113-0). The phage titer after clearing was 6.3 x 10 ¹¹ pfu/ml.

100 μΐ of M-280 paramagnetic Streptavidin beads (Invitrogen, cat#11206D) were washed with 1 ml of PBS-0.05%-Tween-20 (PBS-T) and buffer aspirated. 1 ml of 1% BSA-PBS-0.05% Tween were added to the beads and blocked overnight at 4°C with rotation. The blocking buffer was aspirated, the beads were washed once in 1 ml of PBS and resuspended in 90 ul of PBS.

10 μΐ of a 1 mM solution of biotinylated binding protein partner (SEQ ID NO

65) were added to the blocked beads and incubated at 1 h with rotation to allow for binding of the biotin-group at the N-terminus of the binding protein partner to the

Streptavidin groups on the surface of the beads. After 1 h, 100 ul of a 0.5 mM solution of free D-biotin (Sigma, cat #B4501) was added to saturate any remaining free

Streptavidin binding sites. After an additional 30 min of rotation, the beads were washed twice with 1 ml of PBS/0.05% Tween-20. The washed beads were finally resuspended in 100 ul of PBS/0.05% Tween-20.

100 μΐ of binding protein partner-coated and blocked beads were incubated with

6.6 x 10 ¹⁰ pfu of cleared T7 library in PBS/0.05% Tween-20 in a volume of 600 μΐ.

Phage and beads were incubated for 3 h under rotation. The beads were washed three times withl ml of PBS/0.05% Tween-20 and finally resuspended in 88 ul of PBS.

To release captured phage from the beads, 10 μΐ of 10X HRV3C Protease buffer and 2 μΐ of HRV3C protease (EMD Millipore Cat #71493) were added and the reaction was incubated at room temperature for 2 hours under rotation. The supernatant containing the released phage was aspirated and the phage titer determined.

The amplified starting phage library was named T18_Amp, the bead-captured and released phage fraction was named T18_Acapt and the non-captured phage fraction T18_Anoncyc.

Library characterisation by Next Generation sequencing

The starting phage library, T18_Amp and the fractions from the bead binding experiment T18_Acapt and T18_Anoncyc were analysed by 250 bp Illumina paired- end MiSeq. The samples were prepared for sequencing by the 2-step PCR described in the Illumina manual "16S Metagenomic Sequencing Library Preparation Guide". Due to low phage numbers, the complete volume of T18_Acapt was amplified in 2.5 ml of BLT5615 host culture before sequencing.

Amplicon PCR

1 μΐ of each phage fraction was used as a template for Amplicon PCR as described in the Illumina manual. The reactions were carried out with equimolar mixtures of four staggered forward and four staggered reverse primers as shown in SEQ ID NOs: 116-119.

Oligonucleotides were synthesised by Sigma as desalted PCR primers. The PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent Cat#600675). Thermocycling conditions were: 5 min at 95°C + 20 cycles [20 sec at 95°C, 20 sec at 60°C, 1 min at 72°C] + 3 min at 72°C. The reactions were cleaned up by Agencourt AMPure XP PCR Purification beads (Beckman Coulter Cat#A63880) as described by the manufacturer. Index PCR

1 μΐ of each purified Amplicon PCR was used as a template for an Index PCR, carried out as described in the Illumina manual with index primers from the recommended Nextera XT Index Kit (Illumina Cat#FC-131-1001). The PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent Cat#600675). Thermocycling conditions were: 2 min at 95°C + 8 cycles [20 sec at 95°C, 20 sec at 58°C, 1 min at 72°C] + 3 min at 72°C. The reactions were cleaned up by Agencourt AMPure XP PCR Purification beads (Beckman Coulter Cat#A63880) as described by the manufacturer.

Samples were submitted for sequencing at the Australian Genome Research

Facility by 250 bp Illumina paired-end MiSeq. Samples were loaded at 600 K/mm cluster density with 15% PhiX. The quality of the raw data was checked by FastQC. The paired-end sequencing reads were converted into Phylomer DNA and Phylomer peptide sequences by the in-house software PhySeq4NGS.

Results:

Characterisation of T18_Amp

The complexity of the Phylomer peptide library T18 was determined as 1.9 x 10 by phage titering before library amplification; the titer of the amplified library was 3.3 x 10 ¹¹ pfu/ml.

Several library parameters were determined by NGS. As a form of quality control, NGS was used to assess the genome coverage in the library.

TABLE 14 Summary of Genome Coverage of Phylomer Peptide Library T18

Table 14 provides a list of the genomes covered in T18_Amp. Fold-difference: Observed percentage divided by expected percentage.

Expected representation is achieved if fold-difference is between 0.67 and 1.5. Over-representation if fold-difference >1.5; under-representation if fold-difference <0.67.

Table 14 shows that T18_Amp contained all eight archaebacterial genomes that were inserted into the library. For library cloning, the genomic fragments were pooled according to genome sizes to achieve equal coverage of genomic loci resulting in different expected percentage values for each genome. The observed proportion of genomic fragments from Methanocaldococcus jannaschii in the T18 library was 1.9- fold higher than expected, while the remaining seven genomes were in the expected range with four genomes slightly underrepresented.

Overall, the over/underrepresentation of genomes observed in T18 was in an acceptable range. NGS also allowed the assessment of the library diversity. All peptide sequences of 6 aa and longer were clustered at 80% homology to work out the number of unique peptide sequences (Table 15 below); the copy numbers of all unique sequences are also shown in Table 15. The diversity is reflected in the "diversity number", which is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. The fact that 93% of all unique peptides occurred only once and a diversity number of 0.89 for T18_Amp indicated a very good diversity.

Table 15 Summary of Sequence Diversity in T18 Library

Table 15 provides a summary of the number of unique peptide sequences identified by NGS. The diversity number is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. Copy numbers for unique peptide sequence indicate the percentage of unique sequence that were identified once, twice etc.

Further library parameters are summarised in Table 16 below. All of the general peptide parameters were in the normal range for Phylomer libraries. The mean DNA insert length was determined as 220 bp, which translates into a peptide length of 73 aa. The observed mean peptide length, however, was only 18 aa indicating the presence of non-readthrough inserts caused by stop-codons in the randomly-amplified genomic fragments.

Table 16 Summary of T18 Library Parameters

Table 18 provides a summary of DNA insert lengths, display peptide lengths and percentage of annotated open reading frames (ORF).

The length of each library insert was separately assessed to determine whether the translated peptide was in-frame (IF) or out-of-frame (OOF) with the C-terminal part of the modified binding protein. In the case of stop codons in the reading frame, inserts were classified as non-readthrough (nonRT). The proportions of the three types of library inserts were as follow: 93% of the inserts did not read through into the

C-terminal part of the modified binding protein and were therefore not cyclised. A further 4.7% did read through into the C-terminal part of the modified binding protein but the translation was out-of-frame resulting in a fusion protein that did not function as a modified binding protein and did therefore not cyclise the peptide. Only 2.3% of the Phylomers were classified as readthrough in-frame (RTIF), and could therefore express a functional modified binding protein and display the Phylomer peptide in a cyclised form. The bead binding experiment described below was devised to assess the proportion of actual modified binding proteins with cyclised Phylomers in T18_Amp.

Bead binding experiments

A sample of amplified T18_Amp library was subjected to a binding experiment with immobilised synthetic binding protein partner in order to gain insight into the actual proportion of modified binding proteins with cyclised Phylomers in the library. Only fully functional modified binding proteins with cyclised Phylomers were capable of binding to immobilised binding protein partner, while all other phage clones should remain unbound (as schematically illustrated in Figure 18).

The experiment was carried out with 2.3 x 10 ¹⁰ pfu of phage library and a high amount of binding protein partner immobilised on magnetic beads that should have theoretically allowed the capture of 1000 fold more cyclised phage. Phage titering of the capture fraction and the supernatant after the capture experiment (Table 17) showed that very low numbers of phage equivalent to 0.02% of the phage input were captured instead of the expected 2.3%. This indicates that the proportion of modified binding protein displaying cyclised Phylomers is lower than expected. However, similar, earlier magnetic capture experiments have been shown to be inefficient capture due to steric hindrance preventing quantitative binding compounded by incomplete protease release of captured phage from the beads, which is shown by the fact that phage numbers in T18_Anoncyc and T18_Acapt did not add up to 100%. In spite of this shortcoming, all the phage fractions were analysed by NGS to assess whether there were qualititative differences between T18_Anoncyc and and T18_Acapt.

Table 17 Summary of T18 Library Bead Capture Enrichment

Table 17 provides phage titer results of bead binding experiment. Phage numbers in T18_Anoncyc (supernatant after bead capture) and T18_Acapt (bead- captured fraction) should have added up to 100%.

Comparison of the NGS-derived parameters of T18_Amp, T18_Acycl and

T18_Acapt revealed that the proportion of RTIF clones was one of the main differences between the two fractions obtained by the bead binding experiment (summarised in Figure 17).

Compared to the starting library, the proportion of RTIF phage clones in T18_Acapt was increased 5.3fold (from 2.3 to 12.2%), while the proportion of RTIF phage clones in T18_Anoncyc was equal to that of the starting library. This suggests that the bead-immobilised binding protein partner had preferentially bound phage clones with modified binding protein displaying cyclised Phylomers.

Analysis of the peptide lengths of RTIF phage clones showed that the peptide length distribution in the bead captured fraction T18_Acapt was clearly biased towards shorter peptide lengths while peptide lengths in the starting library (T18_Amp) and the unbound fraction (T18_Anoncyc) were very similar to each other. The display of smaller peptides in loop 2 of the binding protein is probably favoured because protein folding is less disrupted.

Despite the ineffiencies of the bead binding experiment, the enrichment for shorter peptides in T18_Acapt indicates that a subset of the library phage clones displays functional, modified binding protein with cyclised Phylomers. The proportion of these phage clones is lower than 2% but probably higher than suggested by the bead binding experiment and is best assessed by library screening with a target that requires a cyclic binding partner.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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