FIELD OF THE INVENTION

The present invention relates to peptide ligands, such as bicyclic peptide ligands, specific for transferrin receptor 1 (TfR1). The invention also includes pharmaceutical compositions comprising said peptide ligands and the use of said peptide ligands and pharmaceutical compositions in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat. Rev. Drug. Discov. 7(7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 A ² ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin aVb3 (355 A ² ) (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A ² ; Zhao et al. (2007), J. Struct. Biol. 160(1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MM P-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J. Med. Chem. 41 (11), 1749- 51). The favourable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.

Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example 1 ,T,1"-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en-1-one (TATA) (Heinis eta/.(2014) Angewandte Chemie, International Edition 53(6) 1602-1606).

Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat. Chem. Biol. 5(7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)e- Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule scaffold.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a peptide ligand specific for transferrin receptor 1 (TfR1) which comprises an amino acid sequence which is selected from: C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1 , herein referred to as BCY23180); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW(SEQ ID NO: 3, herein referred to as BCY23182); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, herein referred to as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, herein referred to as BCY23185);

CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, herein referred to as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, herein referred to as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, herein referred to as BCY23188);

CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21 , herein referred to as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, herein referred to as

BCY23201);

CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, herein referred to as

BCY23202);

CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, herein referred to as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, herein referred to as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, herein referred to as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, herein referred to as BCY23206);

CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, herein referred to as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, herein referred to as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, herein referred to as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31 , herein referred to as BCY23210);

CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, herein referred to as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, herein referred to as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, herein referred to as BCY23217); CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, herein referred to as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, herein referred to as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, herein referred to as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, herein referred to as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, herein referred to as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, herein referred to as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41 , herein referred to as BCY23224); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, herein referred to as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, herein referred to as BCY23231); and

CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, herein referred to as BCY23515), wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy- phenylalanine, Gia represents L-y-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homo-tyrosine, 3HyV represents 3-hydroxy-L- valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4- carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, tBuGly represents t-butyl-glycine.

According to a further aspect of the invention, there is provided a bicyclic peptide ligand which comprises a peptide ligand as defined herein wherein the first, second and third cysteine residues within said peptide ligands are covalently bonded to a molecular scaffold such that two polypeptide loops are formed on said molecular scaffold.

According to a yet further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or bicyclic peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided a peptide ligand, bicyclic peptide ligand or pharmaceutical composition as defined herein for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent. DETAILED DESCRIPTION OF THE INVENTION

Peptide Ligands

According to a first aspect of the invention, there is provided a peptide ligand specific for transferrin receptor 1 (TfR1) which comprises an amino acid sequence which is selected from: C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1 , herein referred to as BCY23180);

C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW(SEQ ID NO: 3, herein referred to as BCY23182);

CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, herein referred to as BCY23184);

CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, herein referred to as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, herein referred to as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, herein referred to as BCY23187);

CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, herein referred to as BCY23188);

CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21 , herein referred to as BCY23200);

CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, herein referred to as

BCY23201);

CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, herein referred to as

BCY23202);

CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, herein referred to as BCY23203);

CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, herein referred to as BCY23204);

CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, herein referred to as BCY23205);

CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, herein referred to as BCY23206);

CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, herein referred to as BCY23207);

CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, herein referred to as BCY23208);

CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, herein referred to as

BCY23209);

CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31 , herein referred to as

BCY23210);

CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, herein referred to as BCY23211);

CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, herein referred to as BCY23216);

CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, herein referred to as BCY23217);

CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, herein referred to as BCY23218);

CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, herein referred to as BCY23219);

CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, herein referred to as BCY23220);

CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, herein referred to as BCY23221);

CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, herein referred to as BCY23222);

CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, herein referred to as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41 , herein referred to as BCY23224); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, herein referred to as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, herein referred to as BCY23231); and

CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, herein referred to as BCY23515), or a pharmaceutically acceptable salt of said peptide ligand thereof, wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gia represents L-y-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homo-tyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, tBuGly represents t-butyl-glycine.

In a further aspect of the invention which may be mentioned, there is provided a peptide ligand specific for transferrin receptor 1 (TfR1) which comprises an amino acid sequence which is selected from:

C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1 , herein referred to as BCY23180); C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 2, herein referred to as BCY23181); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW(SEQ ID NO: 3, herein referred to as BCY23182); CP[Oxa]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 4, herein referred to as BCY23183); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, herein referred to as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, herein referred to as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, herein referred to as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, herein referred to as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, herein referred to as BCY23188); CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (SEQ ID NO: 10, herein referred to as BCY23189); CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (SEQ ID NO: 11 , herein referred to as BCY23190); CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (SEQ ID NO: 12, herein referred to as BCY23191); CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (SEQ ID NO: 13, herein referred to as BCY23192); CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (SEQ ID NO: 14, herein referred to as BCY23193); CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (SEQ ID NO: 15, herein referred to as BCY23194); CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (SEQ ID NO: 16, herein referred to as BCY23195); CP[HyP]DAYLSC[tBuGly]SYCEPW (SEQ ID NO: 17, herein referred to as BCY23196); CP[HyP]DAYLDC[tBuGly]SYCEPW (SEQ ID NO: 18, herein referred to as BCY23197); CP[HyP]DAYLYC[tBuGly]SYCEPW (SEQ ID NO: 19, herein referred to as BCY23198); CP[HyP]DAYLNC[tBuGly]SYCEPW (SEQ ID NO: 20, herein referred to as BCY23199);

CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21 , herein referred to as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, herein referred to as

BCY23201);

CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, herein referred to as

BCY23202);

CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, herein referred to as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, herein referred to as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, herein referred to as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, herein referred to as BCY23206);

CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, herein referred to as BCY23207);

CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, herein referred to as BCY23208);

CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, herein referred to as

BCY23209);

CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, herein referred to as

BCY23210);

CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, herein referred to as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, herein referred to as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, herein referred to as BCY23217); CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, herein referred to as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, herein referred to as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, herein referred to as BCY23220);

CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, herein referred to as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, herein referred to as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, herein referred to as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, herein referred to as BCY23224); CP[HyP]DAYLGC[tBuGly]TYCEPW (SEQ ID NO: 42, herein referred to as BCY23225);

CP[HyP]DAYLGC[tBuGly]DYCEPW (SEQ ID NO: 43, herein referred to as BCY23226); CP[HyP]DAYLGC[tBuGly]EYCEPW (SEQ ID NO: 44, herein referred to as BCY23227); CP[HyP]DAYLGC[tBuGly]NYCEPW (SEQ ID NO: 45, herein referred to as BCY23228); CP[HyP]DAYLGC[tBuGly]QYCEPW (SEQ ID NO: 46, herein referred to as BCY23229); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, herein referred to as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, herein referred to as BCY23231);

CP[HyP]DAYLGCYSYCEPW (SEQ ID NO: 49, herein referred to as BCY23514); and CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, herein referred to as BCY23515), wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy- phenylalanine, Gia represents L-y-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homo-tyrosine, 3HyV represents 3-hydroxy-L- valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4- carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, tBuGly represents t-butyl-glycine.

It will be appreciated that each of the peptide ligands of the invention comprise an N-terminal acetyl group and a C-terminal CONH2 group.

It will also be appreciated that the term “specific for TfR1” refers to the ability of the peptide ligand to bind to transferrin receptor 1 (TfR1). It will also be appreciated that the peptide ligand will have a differing affect upon TfR1 depending on the precise epitope of binding. For example, the affect will either be inhibitory (i.e. the peptide ligand impedes/inhibits the binding of transferrin to TfR1) or non-inhibitory (i.e. the peptide ligand does not impede/inhibit the binding of transferrin to TfR1.

In a further embodiment, the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium or ammonium salt.

Bicyclic Peptide Ligands

According to a further aspect of the invention, there is provided a bicyclic peptide ligand which comprises a peptide ligand as defined herein wherein the first, second and third cysteine residues within said peptide ligands are covalently bonded to a molecular scaffold such that two polypeptide loops are formed on said molecular scaffold.

In one embodiment, the molecular scaffold is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues.

For the purpose of this description, bicyclic peptides are assumed to be cyclised with TATB to yield a tri-substituted structure. However, as will be clear from the descriptions of the invention presented herein, cyclisation may be performed with any suitable molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed. Cyclisation occurs on the first, second and third cysteine residues, respectively.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sam brook et a/., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel etal., Short Protocols in Molecular Biology (1999) 4 ^th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Numbering

When referring to amino acid residue positions within the peptides of the invention, cysteine residues are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within the peptides of the invention is referred to as below: C[HyP]i[HyP]2D3A ₄ Y5L6G7C[tBuGly]8S9YioCEiiPi2Wi3 (SEQ ID NO: 1).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal biotin-G-Sars tail would be denoted as:

[Biot]-G-[Sar ₅ ]-A-(SEQ ID NO: X).

Inversed Peptide Sequences

In light of the disclosure in Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus become C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

Peptide Ligand Definition

A peptide ligand, as referred to herein, refers to a peptide, peptidic or peptidomimetic covalently bound to a molecular scaffold. Typically, such peptides, peptidics or peptidomimetics comprise a peptide having natural or non-natural amino acids, two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide, peptidic or peptidomimetic is bound to the scaffold. In the present case, the peptides, peptidics or peptidomimetics comprise at least three cysteine residues, and form at least two loops on the scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

- Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamics and pharmacokinetic evaluation;

- Protease stability. Bicyclic peptide ligands should in most circumstances demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicyclic peptide lead candidate can be developed in animal models as well as administered with confidence to humans;

- Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra/inter-molecular H-bonding, which is important for formulation and absorption purposes; and

- An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide with short or prolonged in vivo exposure times for the management of either chronic or acute disease states. The optimal exposure time will be governed by the requirement for sustained exposure (for maximal therapeutic efficiency) versus the requirement for short exposure times to minimise toxicological effects arising from sustained exposure to the agent.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1 ,2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1 ,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO'), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH ₄ ⁺ ) and substituted ammonium ions (e.g. NHsR ⁺ , NH2R2 ⁺ , NHRs ⁺ , NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CHs) ₄ ⁺ .

Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with one or more replacement amino acids, such as an alanine, replacement of one or more L- amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids; and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.

In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal residue is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide. In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine and/or the C-terminal cysteine.

In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise p-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines, such as D-alanines. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

- Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved;

- Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

- Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upon target binding and introducing additional cyclisations in the molecule for identical reasons.

(for reviews see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418). Isotopic Variations

The present invention includes all pharmaceutically acceptable (radio)isotope-labelled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ² H (D) and ³ H (T), carbon, such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine, such as ³⁶ CI, fluorine, such as ¹⁸ F, iodine, such as ¹²³ l, ¹²⁵ l and ¹³¹ l, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus, such as ³² P, sulphur, such as S, copper, such as ⁶⁴ Cu, gallium, such as ⁶⁷ Ga or ⁶⁸ Ga, yttrium, such as ⁹⁰ Y and lutetium, such as ¹⁷⁷ Lu, and Bismuth, such as ²¹³ Bi.

Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³ H (T), and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy. Isotopically-labelled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

Molecular Scaffold

In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. References herein to “non-aromatic molecular scaffold” refers to any molecular scaffold as defined herein which does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.

The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

In one embodiment, the molecular scaffold is 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en- 1-one (also known as triacryloylhexahydro-s-triazine (TATA):

TATA.

Thus, following cyclisation with the bicyclic peptides of the invention on the three cysteine residues, the molecular scaffold forms a tri-substituted 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5- triyl)tripropan-1-one derivative of TATA having the following structure: wherein * denotes the point of attachment of the three cysteine residues. In an alternative embodiment, the molecular scaffold is 1 ,3,5-tris(bromoacetyl) hexahydro-1 , 3,5-triazine (TATB):

TATB.

Thus, following cyclisation with the bicyclic peptides of the invention on the cysteine residues, the molecular scaffold forms a tri-substituted 1 ,3,5-tris(bromoacetyl) hexahydro-1 , 3,5-triazine derivative of TATB having the following structure: wherein * denotes the point of attachment of the three cysteine residues.

Synthesis

The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al. (supra).

Thus, the invention also relates to the manufacture of polypeptides or conjugates selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide/conjugate made by chemical synthesis.

Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively, additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91 (26): 12544-8 or in Hikari et al. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA or TATB) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N- or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulphide-linked bicyclic peptide-peptide conjugate.

Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically- acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition). The peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Further examples of other agents which may be administered separately or in conjunction with the peptide ligands of the invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic and anti-thrombotic factors. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered intravenously. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing a peptide ligand according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as transferrin receptor 1 (TfR1) binding agents. According to a further aspect of the invention, there is provided a peptide ligand or pharmaceutical composition as defined herein for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

Transferrins are glycoproteins found in vertebrates which bind to and consequently mediate the transport of Iron (Fe) through blood plasma. It is produced in the liver and contains binding sites for two Fe ³⁺ atoms. Human transferrin is encoded by the TF gene and produced as a 76 kDa glycoprotein.

Transferrin glycoproteins bind iron tightly, but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate of turnover (25 mg/24 h). Transferrin has a molecular weight of around 80 kDa and contains two specific high-affinity Fe(lll) binding sites. The affinity of transferrin for Fe(lll) is extremely high (association constant is 10 ² ° M" ¹ at pH 7.4) but decreases progressively with decreasing pH below neutrality. Transferrins are not limited to only binding to iron but also to different metal ions. These glycoproteins are located in various bodily fluids of vertebrates. When not bound to iron, transferrin is known as "apotransferrin".

In one embodiment, the transferrin is mammalian transferrin. In a further embodiment, the mammalian transferrin is human transferrin. In one embodiment, the human transferrin is human transferrin receptor 1 (TfR1 ; also known as CD71). t will be appreciated that TfR1 binding peptides may be useful in the treatment of neurological disorders. Examples of such neurological disorders include but are not limited to: a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioural disorder, and CNS inflammation.

In one embodiment, the neurological disorder is in a human subject. It will be appreciated that the dose amount and/or frequency of administration is modulated to reduce the concentration of peptide ligand to which the red blood cells are exposed. In a further embodiment, the treatment further comprises the step of monitoring the human subject for depletion of red blood cells.

References herein to the term "prevention" involves administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.

Transferrin receptor 1 (TfR1) is an extensively studied model receptor-ligand system and has provided considerable insight into the cellular properties and mechanisms of nutrient/scavenger receptor cargo internalization and endocytic sorting (Qian et al (2002) Pharmacological Reviews 54(4), 561-587). TfR1 is known to undergo constitutive endocytosis and recycling to the plasma membrane and possesses pH-dependent ligand binding to enable proper sorting of endocytosed cargo. Anti-TfR1 antibodies have previously been believed to be the primary agents for TfR1 targeting of oligonucleotide therapeutics, however, the present Tfr1 binding peptide ligands of the invention have the potential for demonstrating efficient and profound knockdown of gene expression in skeletal and cardiac muscle via systemically delivered TfR1 -Bicyclic Peptide-siRNA conjugates.

Thus, in light of this mechanism it is believed that the peptide ligands of the invention may find utility as tissue delivery complexes, such as delivery of the Tfr1-peptide ligand-payload (i.e. siRNA) complex to tissue cells, in particular muscle cells. Thus, according to a further aspect of the invention there is provided a tissue delivery complex which comprises a peptide ligand of the invention bound to TfR1 in combination with a payload, such as another peptide, small molecule drug or oligonucleotide, in particular siRNA.

Said tissue delivery complexes therefore find utility in the treatment of musculoskeletal disorders. Examples of suitable musculoskeletal disorders include, but are not limited, to: 12q14 microdeletion syndrome 2q37 deletion syndrome 3M syndrome

Absence of Tibia

Absence of tibia with polydactyly

Absent patella

Acheiropody

Achondrogenesis type 1A - See Achondrogenesis

Achondrogenesis type 1 B - See Achondrogenesis

Achondrogenesis type 2 - See Achondrogenesis

Achondroplasia

Acro-pectoro-renal field defect

Acrocallosal syndrome, Schinzel type

Acrocapitofemoral dysplasia

Acrocephalopolydactyly

Acrodysostosis

Acrodysplasia scoliosis

Acrofacial dysostosis Catania type

Acrofacial dysostosis Palagonia type

Acrofacial dysostosis Rodriguez type

Acrofrontofacionasal dysostosis syndrome

Acromelic frontonasal dysostosis

Acromesomelic dysplasia

Acromesomelic dysplasia Hunter Thompson type

Acromesomelic dysplasia Maroteaux type

Acromicric dysplasia

Acroosteolysis dominant type

Acropectoral syndrome

Acropectorovertebral dysplasia F form

Acute febrile neutrophilic dermatosis Adactylia unilateral

Adams-Oliver syndrome

Adenosine Deaminase 2 deficiency

ADULT syndrome

Adult-onset Still's disease

Aicardi-Goutieres syndrome

Al Gazali Sabrinathan Nair syndrome

Allain-Babin-Demarquez syndrome

Alpha-mannosidosis

Amyotrophy, neurogenic scapuloperoneal, New England type

Anauxetic dysplasia

Angel shaped phalangoepiphyseal dysplasia

Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome

Ankylosing spondylitis - Not a rare disease

Ankylosing vertebral hyperostosis with tylosis

Anonychia-onychodystrophy with hypoplasia or absence of distal phalanges

Antley Bixler syndrome

Apert syndrome

Arthrogryposis multiplex congenita

Arts syndrome

Aspartylglycosaminuria

Atelosteogenesis type 1

Atelosteogenesis type 2

Atelosteogenesis type 3

Auralcephalosyndactyly

Auriculo-condylar syndrome

Auriculoosteodysplasia

Autosomal dominant spondyloepiphyseal dysplasia tarda

Autosomal recessive early-onset inflammatory bowel disease

Autosomal recessive protein C deficiency

Axial osteomalacia

Axial spondylometaphyseal dysplasia

Baby rattle pelvic dysplasia

Baller-Gerold syndrome

Banki syndrome

Beare-Stevenson cutis gyrata syndrome

Behget disease Benallegue Lacete syndrome

Bethlem myopathy

Beukes familial hip dysplasia

Blau syndrome

Blount disease

BOD syndrome

Bone dysplasia Azouz type

Bone dysplasia lethal Holmgren type

Boomerang dysplasia

Bowing of legs, anterior with dwarfism

Brachycephalofrontonasal dysplasia

Brachydactylous dwarfism Mseleni type

Brachydactyly elbow wrist dysplasia

Brachydactyly long thumb type

Brachydactyly Mononen type

Brachydactyly type A1

Brachydactyly type A2

Brachydactyly type A4

Brachydactyly type A5

Brachydactyly type A6

Brachydactyly type A7

Brachydactyly type B

Brachydactyly type C

Brachydactyly type E

Brachydactyly types B and E combined

Brachyolmia type 3

Branchial arch syndrome X-linked

Brody myopathy

Bruck syndrome 1

Buschke-Ollendorff syndrome

C syndrome

Caffey disease

Campomelia Cumming type

Campomelic dysplasia

Camptobrachydactyly

Camptodactyly arthropathy coxa vara pericarditis syndrome

Camptodactyly syndrome Guadalajara type 2 Camptodactyly, tall stature, and hearing loss syndrome

Camurati-Engelmann disease

Cantu syndrome

Carpenter syndrome

Carpotarsal osteochondromatosis

Cartilage-hair hypoplasia

Catel Manzke syndrome

Cerebellar hypoplasia with endosteal sclerosis

Cerebro-costo-mandibular syndrome

Cervical dystonia

Charlie M syndrome

Cherubism

CHILD syndrome

Childhood hypophosphatasia

Chondrocalcinosis 2

Chondrodysplasia Blomstrand type

Chondrodysplasia punctata 1 , X-linked recessive

Chondrodysplasia punctata Sheffield type

Chondrodysplasia with joint dislocations, GPAPP type

Chondrodysplasia, Grebe type

Chondrosarcoma

Chordoma

Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature

Chronic recurrent multifocal osteomyelitis

Cleft hand absent tibia

Cleidocranial dysplasia

Cleidocranial dysplasia recessive form

Cleidorhizomelic syndrome

CLOVES syndrome

Coccygodynia

CODAS syndrome

Coffin-Siris syndrome

COG1-CDG (CDG-llg)

Cole Carpenter syndrome

Collagenopathy type 2 alpha 1

Condensing osteitis of the clavicle

Congenital adrenal hyperplasia due to cytochrome P450 oxidoreductase deficiency Congenital contractural arachnodactyly

Congenital femoral deficiency

Congenital primary aphakia

Congenital radioulnar synostosis

Cornelia de Lange syndrome

Cousin syndrome

Craniodiaphyseal dysplasia

Cranioectodermal dysplasia

Craniofacial dysostosis with diaphyseal hyperplasia

Craniofacial dyssynostosis

Craniofrontonasal dysplasia

Craniometaphyseal dysplasia, autosomal dominant

Craniometaphyseal dysplasia, autosomal recessive type

Craniosynostosis, anal anomalies, and porokeratosis

Craniotelencephalic dysplasia

Crouzon syndrome

Culler-Jones syndrome

Currarino triad

Curry Jones syndrome

Czech dysplasia metatarsal type

Dandy-Walker malformation with postaxial polydactyly

Dandy-Walker malformation with sagittal craniosynostosis and hydrocephalus

Deficiency of interleukin-1 receptor antagonist

Delayed membranous cranial ossification

Dentatorubral-pallidoluysian atrophy

Desbuquois syndrome

Desmosterolosis

Diaphyseal medullary stenosis with malignant fibrous histiocytoma

Diastrophic dysplasia

Dihydropyrimidine dehydrogenase deficiency - Not a rare disease

Dyggve-Melchior-Clausen syndrome

Dyschondrosteosis nephritis

Dysferlinopathy

Dysosteosclerosis

Dysplasia epiphysealis hemimelica

Dyssegmental dysplasia Rolland-Desbuquois type

Dyssegmental dysplasia Silverman-Handmaker type DYT-GNAL

EEC syndrome

EEM syndrome

Ellis-Van Creveld syndrome

Enthesitis-related juvenile idiopathic arthritis

Epidermolysa bullosa simplex with muscular dystrophy

Epiphyseal dysplasia multiple with early-onset diabetes mellitus

Erdheim-Chester disease

Ewing sarcoma

Familial avascular necrosis of the femoral head

Familial cold autoinflammatory syndrome

Familial hypocalciuric hypercalcemia type 1

Familial hypocalciuric hypercalcemia type 2

Familial hypocalciuric hypercalcemia type 3

Familial Mediterranean fever

Familial osteochondritis dissecans

Familial tumoral calcinosis

Fanconi anemia

Feingold syndrome

Felty's syndrome

Femoral facial syndrome

Femur bifid with monodactylous ectrodactyly

Femur fibula ulna syndrome

Fetal thalidomide syndrome

Fibrochondrogenesis

Fibrodysplasia ossificans progressiva

Fibular aplasia ectrodactyly

Fibular aplasia, tibial campomelia, and oligosyndactyly syndrome

Fibular hemimelia

Fibular hypoplasia and complex brachydactyly

Filippi syndrome

Fitzsimmons-Guilbert syndrome

Focal segmental glomerulosclerosis

Frank Ter Haar syndrome

Freiberg's disease

Frontofacionasal dysplasia

Frontometaphyseal dysplasia Frontonasal dysplasia

Frontonasal dysplasia with alopecia and genital anomaly - See Frontonasal dysplasia

Frontonasal dysplasia-severe microphthalmia-severe facial clefting syndrome - See

Frontonasal dysplasia

Frontorhiny - See Frontonasal dysplasia

Fryns Hofkens Fabry syndrome

Fucosidosis

Fuhrmann syndrome

Galactosialidosis

Gaucher disease type 1

Gaucher disease type 3

Geleophysic dwarfism

Genitopatellar syndrome

Genoa syndrome

Genochondromatosis

Geroderma osteodysplastica

Ghosal hematodiaphyseal dysplasia syndrome

Giant cell tumor of bone

GM1 gangliosidosis type 1

GM1 gangliosidosis type 2

GM1 gangliosidosis type 3

Goldenhar disease

Gorham's disease

Gracile bone dysplasia

Grant syndrome

Greenberg dysplasia

Greig cephalopolysyndactyly syndrome

Gurrieri syndrome

Hallermann-Streiff syndrome

Hand foot uterus syndrome

Hanhart syndrome

Heart-hand syndrome, Slovenian type

Heart-hand syndrome, Spanish type

Hemifacial microsomia

Hemifacial myohyperplasia

Hereditary antithrombin deficiency

Hereditary multiple osteochondromas Holt-Oram syndrome

Hunter-McAlpine syndrome

Hurler syndrome

Hurler-Scheie syndrome

Hyaline fibromatosis syndrome

Hyper-lgD syndrome

Hyperostosis corticalis generalisata

Hyperphosphatemic familial tumoral calcinosis

Hypochondroplasia

Hypophosphatasia

Hypophosphatemic rickets

I cell disease

IMAGe syndrome

Imperforate oropharynx-costo vetebral anomalies

Inclusion body myopathy 3

Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia

Inclusion body myositis

Intellectual disability-spasticity-ectrodactyly syndrome

Iridogoniodysgenesis type 1

I VIC syndrome

Jackson-Weiss syndrome

Jansen type metaphyseal chondrodysplasia

Jeune syndrome

Johnson Munson syndrome

Juvenile dermatomyositis

Juvenile osteoporosis

Juvenile Paget disease

Kaplan Plauchu Fitch syndrome

Kenny-Caffey syndrome type 1

Kenny-Caffey syndrome type 2

Keutel syndrome

Kienbock's disease

Kleiner Holmes syndrome

Klippel Feil syndrome

Klippel-Trenaunay syndrome

Kniest dysplasia

Kniest like dysplasia lethal Kohler disease

Kyphomelic dysplasia

Lacrimo-auriculo-dento-digital syndrome

Lambdoid synostosis

Lambert Eaton myasthenic syndrome

Langer mesomelic dysplasia

Larsen syndrome

Lateral meningocele syndrome

Laurin-Sandrow syndrome

Legg-Calve-Perthes disease

Lenz Majewski hyperostotic dwarfism

Leri pleonosteosis

Leri Weill dyschondrosteosis

Lethal chondrodysplasia Moerman type

Lethal chondrodysplasia Seller type

Levator syndrome

Limb-girdle muscular dystrophy type 1A

Limb-girdle muscular dystrophy type 2A

Limb-girdle muscular dystrophy type 2B

Limb-girdle muscular dystrophy type 2E

Limb-girdle muscular dystrophy type 2F

Limb-girdle muscular dystrophy type 2H

Limb-girdle muscular dystrophy, type 2C

Limb-girdle muscular dystrophy, type 2D

Limb-mammary syndrome

Loeys-Dietz syndrome

Lowry Maclean syndrome

Lowry Wood syndrome

Macrophagic myofascitis

Maffucci syndrome

MAGIC syndrome

Majeed syndrome

Mandibuloacral dysplasia with type A lipodystrophy

Mandibuloacral dysplasia with type B lipodystrophy

Mandibulofacial dysostosis with microcephaly

Mannosidosis, beta A, lysosomal

Marshall syndrome Marshall-Smith syndrome

McCune-Albright syndrome

Meckel syndrome

Median cleft of upper lip with polyps of facial skin and nasal mucosa

Meier-Gorlin syndrome

Melnick-Needles syndrome

Melorheostosis

Melorheostosis with osteopoikilosis

Mesomelia-synostoses syndrome

Mesomelic dwarfism cleft palate camptodactyly

Mesomelic dysplasia Kantaputra type

Mesomelic dysplasia Savarirayan type

Metacarpals 4 and 5 fusion

Metachondromatosis

Metaphyseal acroscyphodysplasia

Metaphyseal chondrodysplasia Schmid type

Metaphyseal chondrodysplasia Spahr type

Metaphyseal dysostosis-intellectual disability-conductive deafness syndrome

Metaphyseal dysplasia maxillary hypoplasia brachydactyly

Metaphyseal dysplasia without hypotrichosis

Metatropic dysplasia

Mevalonic aciduria

Microcephalic osteodysplastic primordial dwarfism type 1

Microcephalic osteodysplastic primordial dwarfism type 2

Microcephalic primordial dwarfism Toriello type

Microsomia hemifacial radial defects

Miller syndrome

Minicore myopathy with external ophthalmoplegia

Monomelic amyotrophy

Muckle- Wells syndrome

Mucolipidosis III alpha/beta

Mucolipidosis type 4

Mucopolysaccharidosis type III

Mucopolysaccharidosis type II IA

Mucopolysaccharidosis type II I B

Mucopolysaccharidosis type NIC

Mucopolysaccharidosis type HID Mucopolysaccharidosis type IV

Mucopolysaccharidosis type IVA

Mucopolysaccharidosis type VII

Muenke Syndrome

Multicentric carpotarsal osteolysis syndrome

Multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia 2

Multiple sulfatase deficiency

Multiple synostoses syndrome 1

Multiple system atrophy

Muscular dystrophy

Muscular dystrophy, congenital, megaconial type

MYH7-related scapuloperoneal myopathy

Myhre syndrome

Myosinopathies

Myostatin-related muscle hypertrophy

Myotonic dystrophy

Myotonic dystrophy type 2

Nager acrofacial dysostosis

Nail-patella syndrome

Nakajo Nishimura syndrome

Neonatal Onset Multisystem Inflammatory disease

Neonatal severe hyperparathyroidism

Nestor-guillermo progeria syndrome

Neurofibromatosis type 1

Nievergelt syndrome

Normophosphatemic familial tumoral calcinosis

Occipital horn syndrome

Oculoauriculofrontonasal syndrome

Oculodentodigital dysplasia

Oculomaxillofacial dysostosis

Oculopharyngeal muscular dystrophy

Oliver syndrome

Ollier disease

Omodysplasia 1

Omodysplasia 2

Opsismodysplasia Orofaciodigital syndrome 1

Orofaciodigital syndrome 10

Orofaciodigital syndrome 11

Orofaciodigital syndrome 2

Orofaciodigital syndrome 3

Orofaciodigital syndrome 4

Orofaciodigital syndrome 5

Orofaciodigital syndrome 6

Orofaciodigital syndrome 8

Orofaciodigital syndrome 9

Oslam syndrome

OSMED Syndrome

Ossification of the posterior longitudinal ligament of the spine - Not a rare disease

Osteoarthropathy of fingers familial

Osteochondritis dissecans

Osteodysplasia familial Anderson type

Osteodysplasty precocious of Danks Mayne and Kozlowski

Osteofibrous dysplasia

Osteogenesis imperfecta type I

Osteogenesis imperfecta type II

Osteogenesis imperfecta type III

Osteogenesis imperfecta type IV

Osteogenesis imperfecta type V

Osteogenesis imperfecta type VI

Osteoglophonic dysplasia

Osteomesopyknosis

Osteopathia striata with cranial sclerosis

Osteopenia and sparse hair

Osteopetrosis autosomal dominant type 1

Osteopetrosis autosomal dominant type 2

Osteopetrosis autosomal recessive 3

Osteopetrosis autosomal recessive 4

Osteopetrosis autosomal recessive 7

Osteopoikilosis and dacryocystitis

Osteoporosis oculocutaneous hypopigmentation syndrome

Osteoporosis-pseudoglioma syndrome

Osteosarcoma Oto-palato-digital syndrome type 1

Oto-palato-digital syndrome type 2

Pachydermoperiostosis

Pacman dysplasia

Pallister-Hall syndrome

Paramyotonia congenita

Parastremmatic dwarfism

PARC syndrome

Parkes Weber syndrome

Patterson-Stevenson-Fontaine syndrome

Pelvic dysplasia arthrogryposis of lower limbs

Periodic fever, aphthous stomatitis, pharyngitis and adenitis

Pfeiffer-type cardiocranial syndrome

Phocomelia ectrodactyly deafness sinus arrhythmia

Pigmented villonodular synovitis

Piriformis syndrome

Platyspondylic lethal skeletal dysplasia Torrance type

Pleoconial myopathy with salt craving

Poland syndrome

Polycystic bone disease

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy

Polydactyly myopia syndrome

Polyostotic osteolytic dysplasia, hereditary expansile

Potassium aggravated myotonia

Preaxial deficiency, postaxial polydactyly and hypospadias

Preaxial polydactyly type 1

Preaxial polydactyly type 2

Preaxial polydactyly type 3

Preaxial polydactyly type 4

Progeria

Progressive osseous heteroplasia

Progressive pseudorheumatoid dysplasia

Protein C deficiency - Not a rare disease

Proteus syndrome

Proximal symphalangism

Pseudoachondroplasia

Pseudoaminopterin syndrome Pseudodiastrophic dysplasia

Pseudohypoparathyroidism type 1A

Pseudohypoparathyroidism type 1C

Pseudopseudohypoparathyroidism

Psoriatic juvenile idiopathic arthritis

Pycnodysostosis

Pyknoachondrogenesis

Pyle disease

Pyoderma gangrenosum

Pyogenic arthritis, pyoderma gangrenosum and acne

Radio-ulnar synostosis type 1 - See Congenital radioulnar synostosis

Radio-ulnar synostosis type 2 - See Congenital radioulnar synostosis

Radioulnar synostosis-microcephaly-scoliosis syndrome

Raine syndrome

Ramon Syndrome

Rapadilino syndrome

Reactive arthritis

Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia

Retinal vasculopathy with cerebral leukodystrophy with systemic manifestations

Rhizomelic chondrodysplasia punctata type 1

Rhizomelic dysplasia Patterson Lowry type

Rhizomelic syndrome

Richieri Costa Da Silva syndrome

Rigid spine syndrome

Roberts syndrome

Saethre-Chotzen syndrome

Salla disease - See Free sialic acid storage disease

SAPHO syndrome

Sarcoidosis - Not a rare disease

Say Meyer syndrome

Say-Field-Coldwell syndrome

Scalp defects postaxial polydactyly

SCARF syndrome

Scheie syndrome

Scheuermann disease

Schimke immunoosseous dysplasia

Schinzel Giedion syndrome Schinzel type phocomelia

Schneckenbecken dysplasia

Schnitzler syndrome

Schwartz Jampel syndrome

Sclerosteosis

Seckel syndrome

Sepiapterin reductase deficiency

Short rib-polydactyly syndrome type 3

Short rib-polydactyly syndrome type 1

Short rib-polydactyly syndrome type 4

Short rib-polydactyly syndrome, Majewski type

Short stature syndrome, Brussels type

Shprintzen-Goldberg craniosynostosis syndrome

Shwachman-Diamond syndrome

Sickle beta thalassemia

Sickle cell anemia

Sillence syndrome

Singleton-Merten syndrome

Slipped capital femoral epiphysis - Not a rare disease

Small patella syndrome

Smith McCort dysplasia

Smith-Lemli-Opitz syndrome

Sotos syndrome

Spheroid body myopathy

Spinal muscular atrophy Ryukyuan type

Spinal muscular atrophy type 1 with congenital bone fractures

Spinal muscular atrophy type 3

Spinal muscular atrophy type 4

Spinal muscular atrophy with respiratory distress 1

Splenogonadal fusion limb defects micrognatia

Split hand foot malformation

Split hand split foot nystagmus

Spondylocamptodactyly

Spondylocarpotarsal synostosis syndrome

Spondylocostal dysostosis 1 - See Spondylocostal dysostosis

Spondylocostal dysostosis 2 - See Spondylocostal dysostosis

Spondylocostal dysostosis 3 - See Spondylocostal dysostosis Spondylocostal dysostosis 4 - See Spondylocostal dysostosis

Spondylocostal dysostosis 5 - See Spondylocostal dysostosis

Spondylocostal dysostosis 6 - See Spondylocostal dysostosis

Spondylodysplastic Ehlers-Danlos syndrome

Spondyloenchondrodysplasia with immune dysregulation

Spondyloepimetaphyseal dysplasia Genevieve type

Spondyloepimetaphyseal dysplasia joint laxity

Spondyloepimetaphyseal dysplasia Matrilin-3 related

Spondyloepimetaphyseal dysplasia Missouri type

Spondyloepimetaphyseal dysplasia Shohat type

Spondyloepimetaphyseal dysplasia Sponastrime type

Spondyloepimetaphyseal dysplasia Strudwick type

Spondyloepimetaphyseal dysplasia with hypotrichosis

Spondyloepimetaphyseal dysplasia with multiple dislocations

Spondyloepimetaphyseal dysplasia X-linked

Spondyloepimetaphyseal dysplasia, Aggrecan type

Spondyloepiphyseal dysplasia congenita

Spondyloepiphyseal dysplasia Maroteaux type

Spondyloepiphyseal dysplasia tarda X-linked

Spondyloepiphyseal dysplasia-brachydactyly and distinctive speech

Spondylometaepiphyseal dysplasia short limb-hand type

Spondylometaphyseal dysplasia Algerian type

Spondylometaphyseal dysplasia corner fracture type

Spondylometaphyseal dysplasia Sedaghatian type

Spondylometaphyseal dysplasia type A4

Spondylometaphyseal dysplasia with cone-rod dystrophy

Spondylometaphyseal dysplasia with dentinogenesis imperfecta

Spondylometaphyseal dysplasia X-linked

Spondylometaphyseal dysplasia, Kozlowski type

Spondyloperipheral dysplasia

Spondylothoracic dysostosis

Sprengel deformity

STAR syndrome

Stiff person syndrome

Stuve-Wiedemann syndrome

Symphalangism with multiple anomalies of hands and feet

Syndactyly Cenani Lenz type Syndactyly type 3

Syndactyly type 5

Syndactyly type 9

Syndactyly-polydactyly-earlobe syndrome

Syngnathia multiple anomalies

Synovial Chondromatosis

Systemic onset juvenile idiopathic arthritis

TAR syndrome

TARP syndrome

Tarsal carpal coalition syndrome

Tarsal tunnel syndrome

Tetra-amelia syndrome

Tetraamelia-multiple malformations syndrome

Tetramelic monodactyly

Thanatophoric dysplasia type 1

Thanatophoric dysplasia type 2

Thoracic dysplasia hydrocephalus syndrome

Thoracolaryngopelvic dysplasia

Tibia absent polydactyly arachnoid cyst

Tietze syndrome

TMEM165-CDG (CDG-llk)

Townes-Brocks syndrome

Treacher Collins syndrome

Tricho-dento-osseous syndrome

Trichohepatoenteric syndrome

Trichorhinophalangeal syndrome type 1

Trichorhinophalangeal syndrome type 2

Trichorhinophalangeal syndrome type 3

Trigonobrachycephaly, bulbous bifid nose, micrognathia, and abnormalities of the hands and feet

Triphalangeal thumbs brachyectrodactyly

Trochlea of the humerus aplasia of

Trochlear dysplasia

Troyer syndrome

Tubular aggregate myopathy

Tumor necrosis factor receptor-associated periodic syndrome

Ulna and fibula, hypoplasia of Ulna hypoplasia-intellectual disability syndrome

Ulna metaphyseal dysplasia syndrome

Ulnar hypoplasia lobster claw deformity of feet

Ulnar-mammary syndrome

Undifferentiated pleomorphic sarcoma

Upington disease

Verloes Bourguignon syndrome

Viljoen Kallis Voges syndrome

Warman Mulliken Hayward syndrome

Weaver syndrome

Weill-Marchesani syndrome

Weissenbacher-Zweymuller syndrome

Weyers acrofacial dysostosis

Wildervanck syndrome

Worth type autosomal dominant osteosclerosis

Wrinkly skin syndrome

X-linked dominant chondrodysplasia punctata 2

X-linked dominant scapuloperoneal myopathy

X-linked hypophosphatemia

X-linked intellectual disability-plagiocephaly syndrome

X-linked skeletal dysplasia-intellectual disability syndrome

Yunis-Varon syndrome

The invention is further described below with reference to the following examples.

EXAMPLES

Materials and Methods

Preparation of Bicyclic Peptide Ligands (General Method)

Bicycle peptides were synthesized on Rink amide resin using standard Fmoc (9- fluorenylmethyloxycarbonyl) solid-phase peptide synthesis, either by manual coupling (for large scale) or using a Biotage Syroll automated peptide synthesizer (for small scale). Following TFA-based cleavage from the resin, peptides were precipitated with diethyl ether and dissolved in 50:50 acetonitrile/water. The crude peptides (at ~1 mM concentration) were then cyclized with 1.3 equiv. of the scaffold, using ammonium bicarbonate (100 mM) as a base. Completion of cyclization was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) or LC-MS. Once complete, the cyclization reaction was quenched using N-acetyl cysteine (10 equiv. with respect to the peptide), and the solutions were lyophilized. The residue was dissolved in an appropriate solvent and purified by RP-HPLC. Peptide fractions of sufficient purity and the correct molecular weight (verified by either MALDI- TOF and HPLC or LC-MS) were pooled and lyophilized. Concentrations were determined by UV absorption using the extinction coefficient at 280 nm, which was based on Trp/Tyr content.

All amino acids, unless noted otherwise, were used in the L-configurations.

BIOLOGICAL DATA

The bicyclic peptide ligands of the invention were tested in the following assay:

1. TfR1 SPR Binding Assay

Biacore experiments may be performed to determine k _a (M' ¹ s ^-1 ), kd (s ^-1 ), KD (nM) values of various peptides binding to TfR1.

Recombinant human and cynomolgus TfR1 were received from Bicycle as Hise-tagged TfR1 (a.a. 89-760) (ACRO Biosystems, CD1-H5243 and TFR-C524a).

For analysis of TfR1 peptide binding, a Biacore T200 or S200 instrument was used utilising a capture/coupling approach with a Cytiva NTA chip at 25°C with 25mM HEPES, 0.1 M NaCI, 0.05% Tween 20 pH 7.4 as the running buffer. Immobilisation was carried out as follows. The chip was pre-equilibrated with an injection of 500mM EDTA (pH 8), before activation with 5mM NiSC . The surface was then activated using standard amine-coupling chemistry. Briefly, the carboxymethyl dextran surface was activated with a 1 :1 ratio of 0.4 M 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M /V-hydroxy succinimide (NHS). The TfR1 protein (human or cynomolgus) was then captured onto the activated surface after dilution into running buffer to 200nM and 250nM respectively. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N (1 :1). Reference surfaces were activated and blocked as above with no TfR1 protein capture. Capture levels were in the range of 1 ,500-5,000 RU dependent upon the individual study Buffer was changed to 25mM HEPES, 0.1 M NaCI, 0.05% Tween 20 pH 7.4 1% DMSO.

A dilution series of test peptides was prepared in this buffer with a top peptide concentration of 5pM and 6 further 2-fold dilutions. The SPR analysis was run at 25°C at a flow rate of 30pl/min with 160 seconds association and 700-800 seconds dissociation. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1 :1 binding model allowing for mass transport effects where appropriate. Table 1 : Results of Human TfR1 SPR Binding Assay