A bicyclic peptide ligand specific for transferrin receptor 1 (TfR1)

Abstract

The present invention relates to a peptide ligand specific for transferrin receptor 1 (TfR1), for example a bicyclic peptide ligand. The present invention also includes a pharmaceutical composition comprising the peptide ligand, and the use of the peptide ligand and the pharmaceutical composition in the prevention, suppression or treatment of diseases or disorders by TfR1-mediated delivery of a therapeutic agent.

Description

Technical Field

[0001] Field of the Invention The present invention relates to peptide ligands specific for transferrin receptor 1 (TfR1), such as bicyclic peptide ligands. The present invention also includes pharmaceutical compositions comprising said peptide ligands, and the use of said peptide ligands and pharmaceutical compositions in the prevention, suppression or treatment of diseases or disorders by TfR1-mediated delivery of therapeutic agents.

Background Art

[0002] Background of the Invention Cyclic peptides are an attractive class of molecules for therapeutic development because they can bind to protein targets with high affinity and specificity. Indeed, several cyclic peptides, such as the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer agent octreotide, have already been successfully used clinically (Driggers et al. (2008), Nat. Rev. Drug. Discov. 7(7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Generally, for example, the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide having an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 (603 Å 2 ; Zhao et al. (2007), J. Struct. Biol. 160(1), 1-10) that binds to urokinase-type plasminogen activator, large cyclic peptides bind to surfaces of several hundred square angstroms.

[0003] Peptide macrocyclic compounds, due to their cyclic arrangement, have lower flexibility compared to linear peptides. As a result, the entropy loss during binding to the target is small, leading to high binding affinity. Also, the reduced flexibility fixes the target-specific conformation, enhancing the binding specificity compared to linear peptides. This effect is demonstrated by the example that a potent and selective inhibitor against matrix metalloproteinase 8 (MMP-8) loses its selectivity compared to other MMPs when it is ring-opened (Cherney et al. (1998), J. Med. Chem. 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more prominent in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0004] Previously, various research teams have conjugated polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen et al. used tris(bromomethyl)benzene and its related molecules to rapidly and quantitatively cyclize multiple peptide loops on a synthetic scaffold for structural mimicry of the protein surface (Timmerman et al. (2005), ChemBioChem). A method for the generation of candidate drug compounds is described, which is generated by linking cysteine-containing polypeptides to a molecular scaffold such as 1,1’,1’’-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606).

[0005] The combinatorial approach based on phage display has been developed to generate and screen a large library of bicyclic peptides against a target of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) was displayed on phage and cyclized by covalently attaching the cysteine side chains to a small scaffold.

Summary of the Invention

[0006] According to a first aspect of the present invention, C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, referred to herein as BCY23180); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, referred to herein as BCY23182); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, referred to herein as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, referred to herein as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, referred to herein as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, referred to herein as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, referred to herein as BCY23188); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, referred to herein as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, referred to herein as BCY23201); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, referred to herein as BCY23202); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, referred to herein as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, referred to herein as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, referred to herein as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, referred to herein as BCY23206); CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, referred to herein as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, referred to herein as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, referred to herein as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, referred to herein as BCY23210); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, referred to herein as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, referred to herein as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, referred to herein as BCY23217); CP[HyP]DAYGC[tBuGly]SYCEPW (SEQ ID NO: 35, referred to herein as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, referred to herein as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, referred to herein as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, referred to herein as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, referred to herein as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, referred to herein as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, referred to herein as BCY23224); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, referred to herein as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, referred to herein as BCY23231); and CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, referred to herein as BCY23515), 〔wherein, Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents t-butyl-glycine.〕 There is provided a peptide ligand specific for transferrin receptor 1 (TfR1), comprising an amino acid sequence selected from the group consisting of

[0007] According to a further aspect of the present invention, there is provided a bicyclic peptide ligand comprising the peptide ligand as defined herein, wherein the first, second and third cysteine residues within the peptide ligand are covalently bonded to a molecular scaffold such that two polypeptide loops are formed on the molecular scaffold.

[0008] According to yet a further aspect of the present invention, there is provided a pharmaceutical composition comprising the peptide ligand or bicyclic peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0009] According to a further aspect of the present invention, there is provided the peptide ligand, bicyclic peptide ligand or pharmaceutical composition as defined herein for use in the prevention, suppression or treatment of a disease or disorder by TfR1-mediated delivery of a therapeutic agent.

Mode for Carrying Out the Invention

[0010] Detailed Description of the Invention Peptide ligand According to a first aspect of the present invention, C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, referred to herein as BCY23180); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, referred to herein as BCY23182); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, referred to herein as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, referred to herein as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, referred to herein as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, referred to herein as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, referred to herein as BCY23188); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, referred to herein as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, referred to herein as BCY23201); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, referred to herein as BCY23202); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, referred to herein as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, referred to herein as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, referred to herein as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, referred to herein as BCY23206); CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, referred to herein as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, referred to herein as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, referred to herein as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, referred to herein as BCY23210); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, referred to herein as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, referred to herein as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, referred to herein as BCY23217); CP[HyP]DAYGC[tBuGly]SYCEPW (SEQ ID NO: 35, referred to herein as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, referred to herein as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, referred to herein as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, referred to herein as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, referred to herein as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, referred to herein as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, referred to herein as BCY23224); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, referred to herein as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, referred to herein as BCY23231); and CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, referred to herein as BCY23515), [Here, Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents t-butyl-glycine.] There is provided a peptide ligand specific for transferrin receptor 1 (TfR1) comprising an amino acid sequence selected from the group consisting of, or a pharmaceutically acceptable salt of said peptide ligand.

[0011] In a further aspect of the invention that may be mentioned, C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, referred to herein as BCY23180); C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 2, referred to herein as BCY23181); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, referred to herein as BCY23182); CP[Oxa]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 4, referred to herein as BCY23183); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, referred to herein as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, referred to herein as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, referred to herein as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, referred to herein as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 9, referred to herein as BCY23188); CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (SEQ ID NO: 10, referred to herein as BCY23189); CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (SEQ ID NO: 11, referred to herein as BCY23190); CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (SEQ ID NO: 12, referred to herein as BCY23191); CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (SEQ ID NO: 13, referred to herein as BCY23192); CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (SEQ ID NO: 14, referred to herein as BCY23193); CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (SEQ ID NO: 15, referred to herein as BCY23194); CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (SEQ ID NO: 16, referred to herein as BCY23195); CP[HyP]DAYLSC[tBuGly]SYCEPW (SEQ ID NO: 17, referred to herein as BCY23196); CP[HyP]DAYLDC[tBuGly]SYCEPW (SEQ ID NO: 18, referred to herein as BCY23197); CP[HyP]DAYLYC[tBuGly]SYCEPW (SEQ ID NO: 19, referred to herein as BCY23198); CP[HyP]DAYLNC[tBuGly]SYCEPW (SEQ ID NO: 20, referred to herein as BCY23199); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, referred to herein as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, referred to herein as BCY23201); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, referred to herein as BCY23202); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, referred to herein as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, referred to herein as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, referred to herein as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, referred to herein as BCY23206); CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, referred to herein as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, referred to herein as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, referred to herein as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, referred to herein as BCY23210); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, referred to herein as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, referred to herein as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, referred to herein as BCY23217); CP[HyP]DAYGC[tBuGly]SYCEPW (SEQ ID NO: 35, referred to herein as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, referred to herein as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, referred to herein as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, referred to herein as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, referred to herein as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, referred to herein as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, referred to herein as BCY23224); CP[HyP]DAYLGC[tBuGly]TYCEPW (SEQ ID NO: 42, referred to herein as BCY23225); CP[HyP]DAYLGC[tBuGly]DYCEPW (SEQ ID NO: 43, referred to herein as BCY23226); CP[HyP]DAYLGC[tBuGly]EYCEPW (SEQ ID NO: 44, referred to herein as BCY23227); CP[HyP]DAYLGC[tBuGly]NYCEPW (SEQ ID NO: 45, referred to herein as BCY23228); CP[HyP]DAYLGC[tBuGly]QYCEPW (SEQ ID NO: 46, referred to herein as BCY23229); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, referred to herein as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, referred to herein as BCY23231); CP[HyP]DAYLGCYSYCEPW (SEQ ID NO: 49, referred to herein as BCY23514); and CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, referred to herein as BCY23515), 〔wherein, Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents t-butyl-glycine.〕 There is provided a peptide ligand specific for transferrin receptor 1 (TfR1) comprising an amino acid sequence selected from:

[0012] Each of the peptide ligands of the present invention is understood to comprise an N-terminal acetyl group and a C-terminal CONH 2 group.

[0013] The term "specific for TfR1" is also understood to refer to the ability of a peptide ligand to bind to transferrin receptor 1 (TfR1). It is also understood that the peptide ligand may have different effects on TfR1 depending on the exact epitope to which it binds. For example, the effect may be inhibitory (i.e., the peptide ligand inhibits / suppresses the binding of transferrin to TfR1) or non-inhibitory (i.e., the peptide ligand does not inhibit / suppress the binding of transferrin to TfR1).

[0014] In a further embodiment, the pharmaceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium or ammonium salt.

[0015] Bicyclic peptide ligand According to a further aspect of the present invention, there is provided a bicyclic peptide ligand comprising a peptide ligand as defined herein, wherein the first, second and third cysteine residues within the peptide ligand are covalently bound to a molecular scaffold such that two polypeptide loops are formed on the molecular scaffold.

[0016] In certain embodiments, the molecular scaffold is

Chemical formula

[0017] For the purposes of this description, it is assumed that the bicyclic peptide is cyclized with TATB to form a trisubstituted structure. However, as is apparent from the description of the invention provided herein, cyclization can be carried out using any suitable molecular scaffold that forms a covalent bond with a reactive group of the polypeptide such that at least two polypeptide loops are formed. Cyclization occurs at the first, second and third cysteine residues, respectively.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, such as the techniques of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard methods incorporated herein by reference are used for molecular biology, genetics and biochemical methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th (see, e.g., ed., John Wiley & Sons, Inc.).

[0019] Numbering When referring to the positions of amino acid residues within the peptides of the present invention, cysteine residues are omitted from the numbering because they are invariant. Thus, the numbering of amino acid residues within the peptides of the present invention is C[HyP] 1 [HyP] 2 D 3 A 4 Y 5 L 6 G 7 C[tBuGly] 8 S 9 Y 10 CE 11 P 12 W 13 (SEQ ID NO: 1) as set forth.

[0020] Molecular form The N-terminal extension or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, the N-terminal biotin-G-Sar 5 tail is [Biot]-G-[Sar 5 -A-(SEQ ID NO: X) is shown as.

[0021] Reverse peptide sequence In view of the disclosure of Nair et al (2003) J Immunol 170(3), 1362-1373, the peptide sequences disclosed herein are expected to be useful in retro-inverso form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus and vice versa), and the stereochemistry is also reversed (i.e., D-amino acids become L-amino acids and vice versa).

[0022] Peptide ligand definition As used herein, the peptide ligand refers to a peptide, peptides or peptidomimetics covalently bound to a molecular scaffold. Typically, such peptides, peptides or peptidomimetics include natural or non-natural amino acids, two or more reactive groups capable of forming a covalent bond with the scaffold (i.e., cysteine residues), and a sequence sandwiched between said reactive groups called a loop sequence because the peptide, peptides or peptidomimetics form a loop when bound to the scaffold. In the case of the present invention, the peptide, peptides or peptidomimetics contain at least three cysteine residues and form at least two loops on the scaffold.

[0023] Advantages of Peptide Ligands Certain bicyclic peptides of the present invention have many advantageous properties that allow them to be considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include the following: - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations; - Protease stability. Bicyclic peptide ligands should ideally exhibit stability against most plasma proteases, epithelial ("membrane-bound") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic peptide lead candidates can be developed in animal models and administered to humans with confidence; - Desirable solubility profile. This is a function of charged residues, the ratio of hydrophilic to hydrophobic residues, and intramolecular / intermolecular hydrogen bonding and is important for formulation and absorption purposes; and - Optimal plasma half-life in vivo. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short or long in vivo exposure times to manage chronic or acute disease states. The optimal exposure time is determined by the need for sustained exposure (to maximize therapeutic efficacy) and the need for short-term exposure to minimize toxicological effects resulting from sustained exposure to the drug.

[0024] Pharmaceutically acceptable salts It is understood that salt forms are within the scope of the present invention and that references to peptide ligands include salt forms of said ligands.

[0025] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods, for example, the 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 form of these compounds with a suitable base or acid in water, an organic solvent, or a mixture of both.

[0026] Acid addition salts (mono- or di-salts) can be formed using a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, valeric acid, acylated amino acids, and salts formed with cation exchange resins, and include mono- or di-salts formed from acids selected from the group consisting of.

[0027] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0028] If the compound is anionic or has a functional group that can be present anionicly (e.g., COOH can be present as COO - ), it can form a salt with an organic or inorganic base that generates an appropriate cation. Examples of suitable inorganic cations include 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 + , but are not limited thereto. Examples of suitable organic cations include ammonium ions (i.e., NH 4+ ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ), but are not limited thereto. 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(CH 3 ) 4 + .

[0029] If the peptides of the present invention contain amine functional groups, these can form quaternary ammonium salts, for example, by reaction with an alkylating agent by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the present invention.

[0030] Modified derivatives Modified derivatives of the peptide ligands defined herein are understood to be within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (substituting one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substituting one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids, etc.); addition of spacer groups; substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more amino acid residues with one or more amino acids (e.g., substitution with alanine); substitution 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 a bicyclic peptide ligand; substitution of one or more peptide bonds with alternative bonds; modification of the length of the peptide backbone; substitution of hydrogen on the alpha carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid, tyrosine, etc. with appropriate amine, thiol, carboxylic acid, and phenol-reactive reagents to functionalize said amino acids; introduction or substitution of amino acids having orthogonal reactivity suitable for functionalization (e.g., amino acids having an azide group or an alkyne group to enable functionalization at sites having an alkyne or an azide, respectively), and one or more modifications selected therefrom.

[0031] In certain embodiments, the modified derivative comprises an N-terminal modification and / or a C-terminal modification. In further embodiments, the modified derivative comprises an N-terminal modification using appropriate amino-reactive chemistry and / or a C-terminal modification using appropriate carboxy-reactive chemistry. In further embodiments, the N-terminal or C-terminal modification comprises the addition of an effector group including, but not limited to, a cytotoxic agent, a radioactive chelating agent, or a chromophore.

[0032] 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 suitable reagent during peptide synthesis to obtain a molecule with an acetylated N-terminus. This embodiment has the advantage of removing potential recognition sites for aminopeptidases and avoiding the possibility of degradation of the bicyclic peptide.

[0033] In another embodiment, the N-terminal modification comprises the attachment of a molecular spacer group that facilitates the attachment of an effector group and retention of the potency of the bicyclic peptide against its target.

[0034] 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 to obtain a molecule with an amidated C-terminus. This embodiment has the advantage of removing potential recognition sites for carboxypeptidases and reducing the possibility of proteolysis of the bicyclic peptide.

[0035] In one embodiment, the modified derivative comprises substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isosteric / isoelectronic side chains that are not recognized by degradative proteases and do not have any adverse effects on target potency can be selected.

[0036] Alternatively, non-natural amino acids with constrained amino acid side chains can be used such that proteolytic hydrolysis of nearby peptide bonds is structurally and sterically hindered. In particular, these relate to proline analogs, bulky side chains, Cα-disubstituted derivatives (amino isobutyric acid, Aib, etc.), and cyclic amino acids (a simple derivative is amino-cyclopropylcarboxylic acid).

[0037] In certain embodiments, the modified derivative comprises the addition of a spacer group. In further embodiments, the modified derivative comprises the addition of a spacer group to an N-terminal cysteine and / or a C-terminal cysteine.

[0038] In certain embodiments, the modified derivative also comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In further embodiments, the modified derivative comprises the substitution of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0039] In certain embodiments, the modified derivative comprises the substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In another embodiment, the modified derivative comprises the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. An appropriate balance of charged and hydrophobic amino acid residues is an important feature of the bicyclic peptide ligand. For example, hydrophobic amino acid residues affect the degree of binding to plasma proteins and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. These two can combine to affect the half-life, volume of distribution, and exposure of the peptide drug and can be adjusted according to clinical endpoints. Further, (if the peptide drug is administered subcutaneously), an appropriate combination and number of charged and hydrophobic amino acid residues may reduce irritation at the injection site.

[0040] In certain embodiments, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is thought to enhance proteolytic stability by steric hindrance and the properties of D-amino acids that stabilize β-turn structures (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0041] In one embodiment, the modified derivatives include removal of any amino acid residue and substitution with alanine such as D-alanine. This embodiment provides the advantage of identifying important binding residues and removing potential attack sites in proteolysis.

[0042] It should be noted that each of the above modifications can help to intentionally improve the potency or stability of the peptide. Further improvement in potency based on the modification can be achieved through the following mechanisms; - By incorporating a hydrophobic moiety, the hydrophobic effect is utilized to reduce the off-rate and achieve higher affinity; - By incorporating charged groups that utilize long-range ionic interactions, a faster on-rate and higher affinity are realized (see, for example, Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating additional constraints into the peptide, for example, by appropriately constraining the side chains of amino acids so that the entropy loss upon target binding is minimized, or by constraining the backbone torsion angles so that the entropy loss upon target binding is minimized, or by introducing additional cyclization within the molecule for the same reason (for reviews, see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, or Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

[0043] Isotope variants The present invention includes all pharmaceutically acceptable (radioactive) isotope-labeled peptide ligands of the invention, in which 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 normally found in nature, peptide ligands of the invention to which a metal chelate group (referred to as an "effector") is attached and which can retain the associated (radioactive) isotope, and peptide ligands of the invention in which a specific functional group is covalently substituted with an associated (radioactive) isotope or an isotope-labeled functional group.

[0044] Examples of isotopes suitable for inclusion in the peptide ligands of the invention are 2 hydrogen such as 3 H(D) and 11 carbon such as 13 C, 14 C and 36 chlorine such as 18 fluorine such as 123 iodine such as 125 I, 131 I and 13 nitrogen such as 15 N, 15 oxygen such as 17 O, 18 O and 32 phosphorus such as 64 copper such as 67 gallium such as 68 Ga, 90 yttrium such as 177 lutetium such as 213 Lu and

[0045] Certain isotope-labeled peptide ligands of the present invention, such as those incorporating radioisotopes, are useful for studying the tissue distribution of drugs and / or substrates, as well as clinically evaluating the presence and / or absence of targets on diseased tissues. The peptide ligands of the present invention can further have valuable diagnostic properties in that they can be used for the detection or identification of conjugate formation between a labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, luciferase). Tritium, a radioisotope, i.e., 3 3H(T), and carbon-14, i.e., 14 14C are particularly useful for this purpose in terms of being easily incorporated and readily detectable.

[0046] Substitution with heavier isotopes such as deuterium, i.e., 2 2H(D), may be preferred in some situations because it can result in therapeutic advantages due to better metabolic stability, such as an extended in vivo half-life or a reduced required dose.

[0047] 11 13C, 18 18F, 15 15O and 13 15N substitution with positron-emitting isotopes such as can be useful for positron emission tomography (PET) studies to examine target occupancy.

[0048] Isotope-labeled compounds of the peptide ligands of the present invention can generally be produced by conventional techniques known to those skilled in the art or by procedures similar to those described in the following examples using appropriate isotope-labeled reagents in place of previously used unlabeled reagents.

[0049] Molecular scaffold In certain embodiments, the molecular scaffold comprises a non-aromatic molecular scaffold. As used herein, "non-aromatic molecular scaffold" refers to any of the molecular scaffolds defined herein that do not include an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system.

[0050] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0051] As described in the foregoing reference, the molecular scaffold can also be a small molecule, such as a small organic molecule.

[0052] In certain embodiments, the molecular scaffold can be a polymer. In certain embodiments, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates. 。

[0053] In certain embodiments, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0054] The molecular scaffold can include chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0055] In certain embodiments, 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)):

Chemical formula

[0056] Thus, after cyclization with the bicyclic peptide of the present invention at three cysteine residues, the molecular scaffold is

Chemical Formula

[0057] In another embodiment, the molecular scaffold is 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine (TATB):

Chemical Formula

[0058] Thus, after cyclization with the bicyclic peptide of the present invention at cysteine residues, the molecular scaffold is

Chemical Formula

[0059] Synthesis The peptides of the present invention can be synthetically produced by standard techniques followed by reaction with the molecular scaffold in vitro. When doing this, standard chemical reactions can be used. This enables the rapid and large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistries such as those disclosed by Timmerman et al (supra).

[0060] Accordingly, the present invention also relates to the manufacture of a polypeptide or conjugate selected as defined herein, the manufacture including optional further steps as described below. In certain embodiments, these steps are carried out on the final product polypeptide / conjugate produced by chemical synthesis.

[0061] When manufacturing a conjugate or complex, optionally, amino acid residues in the polypeptide of interest can also be substituted.

[0062] The peptide can also be extended, for example, by incorporating another loop, thereby introducing multiple specificities.

[0063] To extend the peptide, simply utilize standard solid-phase or solution-phase chemistry and chemically extend it at the N-terminus, C-terminus, or within the loop of the peptide using orthogonally protected lysine (and analogs). Standard (bio)conjugation techniques can be used to introduce an N-terminus or C-terminus that is activated or activatable. Alternatively, it can be added by fragment condensation or native chemical ligation (e.g., as shown in Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or by an enzyme (e.g., using subtiligase as described in Chang et al Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544-8, or as described in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0064] Alternatively, the peptide may be extended or modified by further bonding via disulfide bonds. This also has the advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA or TATB) can be added during the chemical synthesis of the first peptide to react with three cysteine groups. Then, additional cysteine or thiol is added to the N-terminus or C-terminus of the first peptide, and this cysteine or thiol is allowed to react only with the free cysteine or thiol of the second peptide to form a disulfide-bonded bicyclic peptide-peptide conjugate.

[0065] Furthermore, the addition of other functional or effector groups can be similarly achieved using appropriate chemical methods, coupling at the N-terminus or C-terminus, or coupling via side chains. In certain embodiments, the coupling is performed in such a way as not to inhibit the activity of either object.

[0066] Pharmaceutical composition 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.

[0067] In general, the peptide ligands of the invention are utilized in purified form, together with pharmaceutically suitable excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including physiological saline and / or buffered media. Parenteral solvents include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable adjuvants can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, if necessary to keep the polypeptide conjugate in suspension.

[0068] Intravenous solvents include infusions, nutritional supplements, and electrolyte supplements, such as those based on Ringer's dextrose. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0069] The peptide ligands of the present invention can be used as separately administered components or in combination with other drugs. These can include various immunotherapeutic agents and immunotoxins such as antibodies, antibody fragments, cyclosporine, methotrexate, adriamycin, or cisplatinum. Further examples of other drugs that can be administered separately or in combination with the peptide ligands of the present invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic factors, and antithrombotic factors. The pharmaceutical composition can be a "cocktail" of a combination of various cytotoxic agents or other drugs and the protein ligand of the present invention, or a combination of selected polypeptides according to the present invention having various specificities, whether pooled before administration or not, for example, a combination of polypeptides selected using different target ligands.

[0070] The route of administration of the pharmaceutical composition according to the present invention can be any that are generally known to those skilled in the art. For treatment, the peptide ligands of the present invention can be administered to any patient according to standard techniques. Administration can be carried out in any suitable manner, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, via the pulmonary route, or by direct injection through a catheter as appropriate. Preferably, the pharmaceutical composition according to the present invention is administered intravenously. The dosage and frequency of administration depend on the patient's age, gender, and condition, the co-administration of other drugs, contraindications, and other parameters that the clinician should consider.

[0071] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be used. It is understood by those skilled in the art that lyophilization and reconstitution can result in varying degrees of loss of activity and that levels may need to be adjusted upward to compensate for this.

[0072] Compositions containing the peptide ligands of the present invention or cocktails thereof can be administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, regulation, killing, or other measurable parameter of a selected population of cells is defined as a "therapeutically effective dose." The amount required to achieve this dose varies depending on the severity of the disease, but generally a selected peptide ligand within the range of 0.005 - 5.0 mg per kg of body weight is used, with 0.05 - 2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands of the present invention or cocktails thereof can also be administered at similar or slightly lower doses.

[0073] Compositions containing peptide ligands according to the present invention can be utilized in prophylactic and therapeutic scenarios to assist in the alteration, inactivation, killing, or removal of selected target cell populations in mammals. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to effectively kill, deplete, or otherwise remove target cell populations from aggregates of heterogeneous cells. Blood from a mammal can be bound ex vivo to a selected peptide ligand. Thereby, unwanted cells are killed from the blood or otherwise removed according to standard techniques for return to the mammal.

[0074] Therapeutic use The bicyclic peptides of the present invention have specific utility as transferrin receptor 1 (TfR1) binders. According to a further aspect of the present invention, there are provided peptide ligands or pharmaceutical compositions as defined herein for use in the prevention, suppression or treatment of diseases or disorders by TfR1-mediated delivery of therapeutic agents.

[0075] Transferrin is a glycoprotein found in vertebrates that binds to iron (Fe) in plasma and, as a result, mediates the transport of iron (Fe). Transferrin is produced in the liver and contains binding sites for two Fe 3+ atoms. Human transferrin is encoded by the TF gene and is produced as a 76 kDa glycoprotein.

[0076] The transferrin glycoprotein binds tightly but reversibly to iron. The iron bound to transferrin forms the most rapidly turning over (25 mg / 24 hours) and most important iron pool, although it is less than 0.1% (4 mg) of the total body iron. The molecular weight of transferrin is approximately 80 kDa and it contains two specific high-affinity Fe(III) binding sites. The affinity of transferrin for Fe(III) is extremely high (the association constant at pH 7.4 is 10 20 M -1 ) and decreases gradually as the pH becomes lower than neutral. Transferrin binds not only iron but also various metal ions. These glycoproteins are present in various body fluids of vertebrates. Transferrin not bound to iron is known as "apotransferrin".

[0077] In certain embodiments, the transferrin is mammalian transferrin. In further embodiments, the mammalian transferrin is human transferrin. In certain embodiments, the human transferrin is human transferrin receptor 1 (TfR1; also known as CD71).

[0078] It is understood that the TfR1-binding peptide may be useful in the treatment of neuropathy. Examples of such neuropathies include, but are not limited to, neuropathy disorders, neurodegenerative diseases, cancer, ophthalmic disorders, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, movement disorders, and CNS inflammation.

[0079] In certain embodiments, the neuropathy is in a human subject. It is understood that the dosage and / or frequency of administration are adjusted to lower the concentration of the peptide ligand to which the erythrocytes are exposed. In further embodiments, the treatment further comprises the step of monitoring the human subject for a decrease in erythrocytes.

[0080] As used herein, the term "prevention" includes administration of a protective composition prior to the induction of a disease. "Suppression" refers to administration of a composition after an inducing event and prior to the appearance of clinical symptoms of the disease. "Treatment" includes administration of a protective composition after the symptoms of the disease have manifested.

[0081] Animal model systems are available that can be used to screen for the effectiveness of peptide ligands in protecting against or treating diseases. The use of animal model systems is facilitated by the present invention, which enables the development of polypeptide ligands that can cross-react with human and animal targets and enables the use of animal models.

[0082] Transferrin receptor 1 (TfR1) is a well-studied model receptor-ligand system, and considerable knowledge has been obtained regarding 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 has pH-dependent ligand binding that enables proper sorting of endocytosed cargo. Anti-TfR1 antibodies have been considered the main agents for targeting TfR1 for oligonucleotide therapeutics, but the Tfr1-binding peptide ligand of the present invention has the potential to show efficient and robust knockdown of gene expression in skeletal and cardiac muscle via systemically delivered TfR1-bicyclic peptide-siRNA conjugates.

[0083] Therefore, in light of this mechanism, it is considered that the peptide ligand of the present invention can find utility as a tissue delivery complex, such as the delivery of Tfr1-peptide ligand-payload (i.e., siRNA) complexes to tissue cells, particularly muscle cells.

[0084] Therefore, according to a further aspect of the present invention, there is provided a tissue delivery complex comprising a combination of the peptide ligand of the present invention bound to TfR1 and a payload such as another peptide, small molecule drug or oligonucleotide, particularly siRNA.

[0085] Therefore, the tissue delivery complex finds 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 Tibial defect Tibial defect with polydactyly Patella defect Phocomelia Achondrogenesis type 1A - See Achondrogenesis Achondrogenesis type 1B - See Achondrogenesis Achondrogenesis type 2 - See Achondrogenesis Achondrogenesis imperfecta Acro-pectoro-renal field defect Acrocephalosyndactyly, Schinzel type Acrocapitofemoral dysplasia Acrocephalopolydactyly Acro-osteogenesis imperfecta 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 Acro-osteolysis, dominant form Acropectoral syndrome Acropectorovertebral dysplasia, type F 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 Neurogenic scapulomelic atrophy, New England type Anauxetic osteodysplasia Angel shaped phalangoepiphyseal dysplasia Eyelid fusion-ectodermal defect-cleft lip / palate syndrome Ankylosing spondylitis - not a rare disease Ankylosing hyperostosis of the spine with tyrosis Anonychia with onychodystrophy of the terminal phalanx with aplasia or absence Antley Bixler syndrome Apert syndrome Congenital multiple arthrogryposis Arts syndrome Aspartylglycosaminuria Atelosteogenesis type 1 Atelosteogenesis type 2 Atelosteogenesis type 3 Auralcephalosyndactyly Auriculo-condylar syndrome Auricular osteodysplasia Autosomal dominant spondyloepiphyseal dysplasia Autosomal recessive early-onset inflammatory bowel disease Autosomal recessive protein C deficiency Axial osteomalacia Axial metaphyseal dysplasia Baby rattle pelvic dysplasia Baller-Gerold syndrome Banki syndrome Beare-Stevenson cutis gyrata syndrome Behçet's disease Benallegue Lacete syndrome Bethlem myopathy Beukes familial hip dysplasia Blau syndrome Blount's disease BOD syndrome Bone dysplasia, Azouz type Bone dysplasia, lethal Holmgren type Boomerang dysplasia Flexed legs with nanism Brachycephalofrontonasal dysplasia Brachydactyly-nanism, 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, combination of type B and type E Short trunk, type 3 Branchial arch syndrome, X-linked Brody myopathy Bruck syndrome 1 Buschke - Ollendorff syndrome C syndrome Caffey disease Flexion - deformity of the limbs, Cumming type Flexion - deformity of the limbs with malformation Camptobrachydactyly Camptobrachydactyly with genu valgum and synovitis Camptobrachydactyly syndrome, Guadalajara type 2 Camptobrachydactyly, tall stature, deafness 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 Infantile hypophosphatasia Chondrocalcinosis 2 Chondrodysplasia, Blomstrand type Chondrodysplasia punctata 1, X - linked recessive Chondrodysplasia punctata, Sheffield type Chondrodysplasia with joint dislocation, GPAPP type Chondrodysplasia, Grebe type Chondrosarcoma Chordoma Chronic atypical neutrophilic dermatosis with lipodystrophy and hyperthermia Chronic recurrent multifocal osteomyelitis Cleft hand absent tibia Cleidocranial dysostosis Cleidocranial dysostosis latent Cleidorhizomelic syndrome CLOVES syndrome Coccydynia CODAS syndrome Coffin-Siris syndrome COG1-CDG (CDG-IIg) 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 Craniometaphyseal dysplasia Craniocutaneous dysplasia Craniomaxillofacial dysostosis with metaphyseal overgrowth Craniomaxillofacial synostosis Cranioprefrontonasal dysplasia Craniometaphyseal dysplasia, autosomal dominant Craniometaphyseal dysplasia, autosomal recessive Craniostenosis, 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 premature sagittal suture fusion and hydrocephalus Interleukin-1 receptor antagonist deficiency 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 Chondrodysplasia 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 Multiple epiphyseal dysplasia with early-onset diabetes 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 Bifid femur with syndactyly of the hand and foot Femoral fibular ulnar syndrome Fetal thalidomide syndrome Fibrochondrogenesis imperfecta Progressive osseous heteroplasia Syndactyly of the hand and foot with fibular deficiency Fibular deficiency, tibial bowing and oligodactyly syndrome Fibular hemimelia Fibular hypoplasia and complex brachydactyly Filippi syndrome Fitzsimmons-Guilbert syndrome Focal segmental glomerulosclerosis Frank Ter Haar syndrome Freiberg's disease Anterior craniofacial nasal dysplasia Anterior craniofacial metaphyseal dysplasia Anterior nasal dysplasia Anterior nasal dysplasia with alopecia and genital anomalies - See Anterior nasal dysplasia Anterior nasal dysplasia - Severe microphthalmia - Severe facial cleft syndrome - See Anterior nasal dysplasia Anterior nasal dysplasia Anterior nasal dysplasia (Frontorhiny) - See Anterior nasal 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 with thalassemia 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-uterus syndrome Hanhart syndrome Heart-hand syndrome Slovenian type Heart-hand syndrome Spanish type Hemifacial microsomia Hemifacial myohyperplasia Hereditary antithrombin deficiency Hereditary multiple osteochondroma Holt-Oram syndrome Hunter-McAlpine syndrome Hurler syndrome Hurler-Scheie syndrome Hyaline fibromatosis syndrome Hyper IgD syndrome Hyperostosis corticalis generalisata Hyperphosphatemic familial tumoral calcinosis Hypochondrogenesis Hypophosphatasia Hypophosphatemic rickets I-cell disease IMAGe syndrome Imperforate oropharynx-costo vetebral anomalies Inclusion body myopathy 3 Inclusion body myopathy associated with early-onset Paget's disease and frontotemporal dementia Inclusion body myositis Intellectual disability-spasticity-split hand / foot malformation syndrome Iris angle dysgenesis type 1 IVIC syndrome Jackson-Weiss syndrome Jansen type metaphyseal chondrodysplasia Jeune syndrome Johnson Munson syndrome Juvenile dermatomyositis Juvenile osteoporosis Juvenile Paget's 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 Lethal Kniest-like dysplasia Kohler's disease Kyphomelic dysplasia Lacrimo-auriculo-dento-digital syndrome Lambda suture 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 myofasciitis 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 the upper lip with polyps of the facial skin and nasal mucosa Meier-Gorlin syndrome Melnick-Needles syndrome Melorheostosis Melorheostosis with osteopoikilosis Mesomelia-synostoses syndrome Mesomelic dwarfism, cleft palate, camptodactyly syndrome 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 Dysplastic bone dysplasia Mevalonic aciduria Microcephalic osteodysplasia primordial dwarfism type 1 Microcephalic osteodysplasia primordial dwarfism type 2 Microcephalic primordial dwarfism Toriello type Microsomia hemifacial radial defects Miller syndrome Minicore myopathy with external ophthalmoplegia Monomelic amyotrophy McKusick-Kaufman syndrome Mucolipidosis III alpha / beta Mucolipidosis IV Mucopolysaccharidosis III Mucopolysaccharidosis IIIA Mucopolysaccharidosis IIIB Mucopolysaccharidosis IIIC Mucopolysaccharidosis IIID Mucopolysaccharidosis IV Mucopolysaccharidosis IVA Mucopolysaccharidosis VII Muenke syndrome Multicentric carpal and tarsal osteolysis syndrome Multiple metaphyseal dysplasia Multiple metaphyseal dysplasia 2 Multiple sulfatase deficiency Multiple synostosis syndrome 1 Multiple system atrophy Muscular dystrophy Congenital muscular dystrophy, 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 progeroid syndrome Neurofibromatosis type 1 Nievergelt syndrome Normophosphatemic familial tumoral calcinosis Occipital horn syndrome Oculoauriculovertebral spectrum Oculodentodigital dysplasia Oculomandibulofacial dysplasia Oculopharyngeal muscular dystrophy Oliver syndrome Ollier disease Omodysplasia 1 Omodysplasia 2 Opsismodysplasia Orofaciodigital syndrome 1 Orofaciodigital syndrome 10 Orofaciodigital syndrome 11 Orofacial Digital Syndrome 2 Orofacial Digital Syndrome 3 Orofacial Digital Syndrome 4 Orofacial Digital Syndrome 5 Orofacial Digital Syndrome 6 Orofacial Digital Syndrome 8 Orofacial Digital Syndrome 9 Osler Syndrome OSMED Syndrome Ossification of the posterior longitudinal ligament of the spine - Not a rare disease Familial arthropathy of fingers Osteochondritis Familial osteodysplasia, Anderson type Danks - Main and Kozlowski's early - onset osteodysplasia Fibrous dysplasia of bone 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 Marble bone disease, autosomal dominant type 1 Marble bone disease, autosomal dominant type 2 Marble bone disease, autosomal recessive type 3 Marble bone disease, autosomal recessive type 4 Marble bone disease, autosomal recessive type 7 Osteopathia striata and dacryocystitis Osteoporosis - oculocutaneous hypopigmentation syndrome Osteoporosis - pseudoglioma syndrome Osteosarcoma Oto-palato-digital syndrome type 1 Oto-palato-digital syndrome type 2 Hypertrophic pachydermoperiostosis Pacman dysplasia Pallister-Hall syndrome Congenital paramyotonia Parastremmatic dwarfism PARC syndrome Parkes Weber syndrome Patterson-Stevenson-Fontaine syndrome Pelvic dysplasia and lower limb joint contracture syndrome Periodic fever, aphthous stomatitis, pharyngitis, and adenitis Pfeiffer-type cardiocranial syndrome Short limb, cleft hand and foot, deafness, and atrial arrhythmia Pigmented villonodular synovitis Piriformis syndrome Platyspondylic lethal skeletal dysplasia, Torrance type Pleoconial myopathy with salt craving Poland syndrome Polyostotic bone disease Polyostotic lipomembranous osteodysplasia with sclerosing leukoencephalopathy Polydactyly myopia syndrome Hereditary expansile multiple osteolytic osteodysplasia Potassium-induced 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 heteroplasia Protein C deficiency - not a rare disease Proteus syndrome Proximal interphalangeal joint synostosis Pseudoachondroplasia Pseudoaminopterin syndrome Pseudosacralization of the spine Pseudohypoparathyroidism type 1A Pseudohypoparathyroidism type 1C Pseudopseudohypoparathyroidism Psoriatic juvenile idiopathic arthritis Pycnodysostosis Pyknoachondrogenesis Pyle disease Pyoderma gangrenosum Pyogenic arthritis, pyoderma gangrenosum, and acne Radioulnar synostosis type 1 - see congenital radioulnar synostosis Radioulnar synostosis type 2 - see congenital radioulnar synostosis Radioulnar synostosis, microcephaly, and scoliosis syndrome Rain syndrome Ramon syndrome Rapadilino syndrome Reactive arthritis Renal dysplasia, retinitis pigmentosa, cerebellar ataxia, and skeletal dysplasia Retinal vasculopathy with leukoencephalopathy with systemic manifestations Rhizomelic chondrodysplasia punctata type 1 Rhizomelic dysplasia, Patterson - Lorrie type Rhizomelic syndrome Richieri Costa Da Silva syndrome Rheumatoid spondylitis syndrome Roberts syndrome Saethre - Chotzen syndrome See Mucopolysaccharidosis - Free Sialic Acid Accumulation Disease SAPHO syndrome Sarcoidosis - Not a rare disease Seckel - Meyer syndrome Say - Field - Coldwell syndrome Scalp defect - Postaxial polydactyly SCARF syndrome Scheie syndrome Scheuermann disease Schimke immuno - osteodysplasia Schinzel Giedion syndrome Schinzel - type seal limb disease Snail - like pelvic dysplasia Schnitzler syndrome Schwartz Jampel syndrome Sclerosing osteodysplasia 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 - cell beta - thalassemia Sickle - cell anemia Silence syndrome Singleton - Merten syndrome Femoral head slip - Not a rare disease Patella alta 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 fractures Spinal muscular atrophy type 3 Spinal muscular atrophy type 4 Spinal muscular atrophy 1 with dyspnea Splenic gland fusion, limb defects, micrognathia Syndactyly and cleft hand and foot malformation Syndactyly and cleft hand and foot with nystagmus Spinal digit flexion disorder Spinal carpometacarpal and tarsometatarsal synostosis syndrome Spinal and costal heterotosis 1 - See spinal and costal heterotosis Spinal and costal heterotosis 2 - See spinal and costal heterotosis Spinal and costal heterotosis 3 - See spinal and costal heterotosis Spinal and costal heterotosis 4 - See spinal and costal heterotosis Spinal and costal heterotosis 5 - See spinal and costal heterotosis Spinal and costal heterotosis 6 - See spinal and costal heterotosis Spinal dysplastic Ehlers - Danlos syndrome Spinal chondrodysplasia with immune dysregulation Spondyloepiphyseal dysplasia, Genevieve type Spondyloepiphyseal dysplasia with joint laxity Spondyloepiphyseal dysplasia related to Matrilin - 3 Spondyloepiphyseal dysplasia, Missouri type Spondyloepiphyseal dysplasia, Shohat type Spondyloepiphyseal dysplasia, Sponastrime type Spondyloepiphyseal dysplasia, Strudwick type Spondyloepiphyseal dysplasia with hypotrichosis Spondyloepiphyseal dysplasia with multiple dislocations Spondyloepiphyseal dysplasia, X - linked type Spondyloepiphyseal dysplasia, aggrecan type Congenital spondyloepiphyseal dysplasia Spondyloepiphyseal dysplasia, Maroteaux type X-linked spondyloepiphyseal dysplasia tarda Spondyloepiphyseal dysplasia, brachydactyly, and characteristic speech Spondyloepimetaphyseal dysplasia, short limb - hand type Spondyloepimetaphyseal dysplasia, Algerian type Spondyloepimetaphyseal dysplasia, angular fracture type Spondyloepimetaphyseal dysplasia, Sedaghatian type Spondyloepimetaphyseal dysplasia, type A4 Spondyloepimetaphyseal dysplasia with cone - rod dystrophy Spondyloepimetaphyseal dysplasia with dentinogenesis imperfecta X - linked spondyloepimetaphyseal dysplasia Spondyloepimetaphyseal dysplasia, Kozlowski type Perispinal dysplasia Thoracic spinal heterostosis 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 - preauricular syndrome Gnathosymphalangism - multiple anomalies Synovial chondromatosis Generalized onset juvenile idiopathic arthritis TAR syndrome TARP syndrome Calcaneo - carpal coalition syndrome Tarsal tunnel syndrome Tetra - amelia syndrome Tetra - amelia - multiple congenital anomalies syndrome Ectrodactyly Lethal osteogenesis imperfecta type 1 Lethal osteogenesis imperfecta type 2 Thoracic cage dysplasia - hydrocephalus syndrome Thoracic cage - laryngeal - pelvic dysplasia Tibial deficiency - polydactyly - arachnoid cyst Tietze syndrome TMEM165-CDG (CDG-IIk) Townes-Brocks syndrome Treacher Collins syndrome Hair-tooth-bone syndrome Hair-hepatointestinal syndrome Tricho-rhino-phalangeal syndrome type 1 Tricho-rhino-phalangeal syndrome type 2 Tricho-rhino-phalangeal syndrome type 3 Triangular humeral head, globular nasal cleft, micrognathia, and limb anomalies Triphalangeal thumb and oligodactyly Absence of the trochlea of the humerus Dysplasia of the trochlea Troyer syndrome Tubular aggregate myopathy Tumor necrosis factor-related periodic syndrome Hypoplasia of the ulna and fibula Hypoplasia of the ulna and mental retardation syndrome Metaphyseal dysplasia of the ulna 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 Weber syndrome Weill-Marchesani syndrome Weissenbacher-Zweymuller syndrome Weyers acrofacial dysostosis Wildervank syndrome Warthin type autosomal dominant osteopetrosis Wrinkly skin syndrome X-linked dominant chondrodysplasia punctata 2 X-linked dominant scapuloperoneal myopathy X-linked hypophosphatemia X-linked intellectual disability-torticollis syndrome X-linked skeletal dysplasia-intellectual disability syndrome Yunis-Varon syndrome

[0086] The present invention will be further described below with reference to the following examples.

Examples

[0087] Materials and Methods Production of bicyclic peptide ligand (general method) The bicyclic peptide was synthesized on Rink amide resin using standard Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis with manual coupling (for large scale) or a Biotage SyroII automated peptide synthesizer (for small scale). After cleavage from the resin with TFA, the peptide was precipitated with diethyl ether and dissolved in 50:50 acetonitrile / water. Then, using ammonium bicarbonate (100 mM) as a base, the crude peptide (at approximately 1 mM concentration) was cyclized with 1.3 equivalents of a scaffold. Completion of cyclization was determined by matrix-assisted laser desorption ionization time-of-flight method (MALDI-TOF) or LC-MS. Once cyclization was complete, the cyclization reaction was quenched with N-acetylcysteine (10 equivalents relative to the peptide), and the solution was lyophilized. The residue was dissolved in an appropriate solvent and purified by RP-HPLC. Peptide fractions with sufficient purity and appropriate molecular weight (confirmed by MALDI-TOF and HPLC or LC-MS) were pooled and lyophilized. The concentration was determined by UV absorption using the extinction coefficient at 280 nm based on the Trp / Tyr content.

[0088] Unless otherwise specified, all amino acids were used in the L configuration.

[0089] Biological Data The bicyclic peptide ligand of the present invention was tested in the following assay.

[0090] 1. TfR1 SPR binding assay The k a (M -1 s -1 ) values, k d (s -1 ) values, and K D (nM) values can be determined by performing a Biacore test.

[0091] Recombinant human and cynomolgus monkey TfR1 were obtained from Bicycle as His 6 -tagged TfR1 (amino acids 89 - 760) (ACRO Biosystems, CD1-H5243 and TFR-C524a).

[0092] For the analysis of TfR1 peptide binding, a capture / coupling approach using a Cytiva NTA chip with 25 mM HEPES, 0.1 M NaCl, 0.05% Tween 20 pH 7.4 as the running buffer at 25 °C was utilized using a Biacore T200 or S200 instrument. Immobilization was performed as follows. After injecting 500 mM EDTA (pH 8) to pre-equilibrate the chip, 5 mM NiSO 4It was activated. Subsequently, the surface was activated using standard amine coupling chemistry. Briefly, the carboxymethyldextran surface was activated with 0.4M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1M N-hydroxysuccinimide (NHS) in a 1:1 ratio. Subsequently, the TfR1 protein (human or cynomolgus monkey) was diluted with running buffer to 200 nM and 250 nM respectively, and then captured on the activated surface. The remaining activated groups were blocked by injecting 1M ethanolamine (pH 8.5):HBS-N (1:1) for 7 minutes. The reference surface was activated and blocked in the same manner as above without capturing any TfR1 protein. The capture levels ranged from 1,500 to 5,000 RU depending on the individual study. The buffer was changed to 25 mM HEPES, 0.1M NaCl, 0.05% Tween20 pH 7.4 1% DMSO.

[0093] A dilution series of the test peptide was prepared in this buffer with a maximum peptide concentration of 5 μM, and further two-fold dilutions were performed six times. SPR analysis was carried out at 25 °C, a flow rate of 30 μl / min, 160 seconds of binding, and 700 - 800 seconds of dissociation. The data were corrected for the volume effect excluding DMSO. All data were double-referenced for blank injection and the reference surface using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). The data were fitted using a simple 1:1 binding model considering mass transport effects as necessary.

Table 1

Claims

1. C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 1, referred to herein as BCY23180); C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 2, referred to herein as BCY23181); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 3, referred to herein as BCY23182); CP[Oxa]DAYLGC[tBuGly]SYCEPW (Sequence ID 4, referred to herein as BCY23183); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 5, referred to herein as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (Sequence ID 6, referred to herein as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (Sequence ID 7, referred to herein as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (Sequence ID 8, referred to herein as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (Sequence ID 9, referred to herein as BCY23188); CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (Sequence ID 10, referred to herein as BCY23189); CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (Sequence ID 11, referred to herein as BCY23190); CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (Sequence ID 12, referred to herein as BCY23191); CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (Sequence ID 13, referred to herein as BCY23192); CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (Sequence ID 14, referred to herein as BCY23193); CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (Sequence ID 15, referred to herein as BCY23194); CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (Sequence ID 16, referred to herein as BCY23195); CP[HyP]DAYLSC[tBuGly]SYCEPW (Sequence ID 17, referred to herein as BCY23196); CP[HyP]DAYLDC[tBuGly]SYCEPW (Sequence ID 18, referred to herein as BCY23197); CP[HyP]DAYLYC[tBuGly]SYCEPW (Sequence ID 19, referred to herein as BCY23198); CP[HyP]DAYLNC[tBuGly]SYCEPW (Sequence ID 20, referred to herein as BCY23199); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (Sequence ID 21, referred to herein as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (Sequence ID 22, referred to herein as BCY23201); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (Sequence ID 23, referred to herein as BCY23202); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (Sequence ID 24, referred to herein as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (Sequence ID 25, referred to herein as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (Sequence ID 26, referred to herein as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (Sequence ID 27, referred to herein as BCY23206); CP[HyP]DAYLGC[tBuGly]SYCEPY (Sequence ID 28, referred to herein as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (Sequence ID 29, referred to herein as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (Sequence ID 30, referred to herein as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (Sequence ID 31, referred to herein as BCY23210); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (Sequence ID 32, referred to herein as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (Sequence ID 33, referred to herein as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (Sequence ID 34, referred to herein as BCY23217); CP[HyP]DAYSGC[tBuGly]SYCEPW (Sequence ID 35, referred to herein as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (Sequence ID 36, referred to herein as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (Sequence ID 37, referred to herein as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (Sequence ID 38, referred to herein as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (Sequence ID 39, referred to herein as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (Sequence ID 40, referred to herein as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (Sequence ID 41, referred to herein as BCY23224); CP[HyP]DAYLGC[tBuGly]TYCEPW (Sequence ID 42, referred to herein as BCY23225); CP[HyP]DAYLGC[tBuGly]DYCEPW (Sequence ID 43, referred to herein as BCY23226); CP[HyP]DAYLGC[tBuGly]EYCEPW (Sequence ID 44, referred to herein as BCY23227); CP[HyP]DAYLGC[tBuGly]NYCEPW (Sequence ID 45, referred to herein as BCY23228); CP[HyP]DAYLGC[tBuGly]QYCEPW (Sequence ID 46, referred to herein as BCY23229); CP[HyP]DAYLGC[tBuGly]SYCDPW (Sequence ID 47, referred to herein as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (Sequence ID 48, referred to herein as BCY23231); CP[HyP]DAYLGCYSYCEPW (Sequence ID 49, referred to herein as BCY23514); and CP[HyP]DAYLGC[3HyV]SYCEPW (Sequence ID 50, referred to herein as BCY23515) [Here, Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxyphenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents t-butylglycine.] A peptide ligand specific to transferrin receptor 1 (TfR1) or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence selected from the above.

2. C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 1, referred to herein as BCY23180); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 3, referred to herein as BCY23182); CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (Sequence ID 5, referred to herein as BCY23184); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (Sequence ID 6, referred to herein as BCY23185); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (Sequence ID 7, referred to herein as BCY23186); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (Sequence ID 8, referred to herein as BCY23187); CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (Sequence ID 9, referred to herein as BCY23188); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (Sequence ID 21, referred to herein as BCY23200); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (Sequence ID 22, referred to herein as BCY23201); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (Sequence ID 23, referred to herein as BCY23202); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (Sequence ID 24, referred to herein as BCY23203); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (Sequence ID 25, referred to herein as BCY23204); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (Sequence ID 26, referred to herein as BCY23205); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (Sequence ID 27, referred to herein as BCY23206); CP[HyP]DAYLGC[tBuGly]SYCEPY (Sequence ID 28, referred to herein as BCY23207); CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (Sequence ID 29, referred to herein as BCY23208); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (Sequence ID 30, referred to herein as BCY23209); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (Sequence ID 31, referred to herein as BCY23210); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (Sequence ID 32, referred to herein as BCY23211); CP[HyP]EAYLGC[tBuGly]SYCEPW (Sequence ID 33, referred to herein as BCY23216); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (Sequence ID 34, referred to herein as BCY23217); CP[HyP]DAYSGC[tBuGly]SYCEPW (Sequence ID 35, referred to herein as BCY23218); CP[HyP]DAYTGC[tBuGly]SYCEPW (Sequence ID 36, referred to herein as BCY23219); CP[HyP]DAYDGC[tBuGly]SYCEPW (Sequence ID 37, referred to herein as BCY23220); CP[HyP]DAYEGC[tBuGly]SYCEPW (Sequence ID 38, referred to herein as BCY23221); CP[HyP]DAYNGC[tBuGly]SYCEPW (Sequence ID 39, referred to herein as BCY23222); CP[HyP]DAYQGC[tBuGly]SYCEPW (Sequence ID 40, referred to herein as BCY23223); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (Sequence ID 41, referred to herein as BCY23224); CP[HyP]DAYLGC[tBuGly]SYCDPW (Sequence ID 47, referred to herein as BCY23230); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (Sequence ID 48, referred to herein as BCY23231); and CP[HyP]DAYLGC[3HyV]SYCEPW (Sequence ID 50, referred to herein as BCY23515), [Here, Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxyphenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents t-butylglycine.] A peptide ligand according to claim 1, comprising an amino acid sequence selected from therefor, or a pharmaceutically acceptable salt thereof.

3. The peptide ligand according to claim 1, comprising an N-terminal acetyl group, or a pharmaceutically acceptable salt thereof.

4. The peptide ligand according to claim 1, comprising a C-terminal amide group, or a pharmaceutically acceptable salt thereof.

5. N-terminal acetyl group and C-terminal CONH ２ A peptide ligand according to claim 1, comprising a group, or a pharmaceutically acceptable salt thereof.

6. The peptide ligand or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is selected from free acid or sodium salt, potassium salt, calcium salt or ammonium salt.

7. A bicyclic peptide ligand comprising the peptide ligand described in claim 1 or a pharmaceutically acceptable salt thereof, wherein the first, second, and third cysteine ​​residues in the peptide ligand are covalently bonded to a molecular scaffold such that two polypeptide loops are formed on the molecular scaffold.

8. Molecular scaffolds 【Chemistry 1】 [In the formula, * indicates the binding site of three cysteine ​​residues.] The bicyclic peptide ligand according to claim 7, which is a derivative of TATB having the structure of [the specified structure].

9. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or a bicyclic peptide ligand according to claim 7 or claim 8.

10. A pharmaceutical for preventing, suppressing or treating a disease or disorder through TfR1-mediated delivery of a therapeutic agent, comprising a peptide ligand according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or a bicyclic peptide ligand according to claim 7 or claim 8, or a pharmaceutical composition comprising the peptide ligand or a pharmaceutically acceptable salt thereof or the bicyclic peptide ligand as an active ingredient.

11. A tissue delivery complex comprising a combination of a peptide ligand according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or a bicyclic peptide ligand according to claim 7 or claim 8, bound to TfR1, and a payload.

12. The tissue delivery complex according to claim 11, wherein it is a muscle tissue delivery complex.

13. A pharmaceutical product for treating musculoskeletal disorders, comprising the tissue delivery complex described in claim 11 as an active ingredient.

14. The pharmaceutical product according to claim 13, wherein the tissue delivery complex is a muscle tissue delivery complex.

15. Use in the manufacture of a pharmaceutical for treating a disease or disorder through TfR1-mediated delivery of a therapeutic agent, the peptide ligand or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, or the bicyclic peptide ligand according to claim 7 or claim 8, or a pharmaceutical composition comprising the peptide ligand or a pharmaceutically acceptable salt thereof or the bicyclic peptide ligand.

16. Use of the tissue delivery complex according to claim 11 in the manufacture of a pharmaceutical product for treating musculoskeletal disorders.

17. The use according to claim 16, wherein the tissue delivery complex is a muscle tissue delivery complex.

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