Transferrin receptor 1 (TfR1) specific bicyclic peptide ligands

Abstract

The present invention relates to peptide ligands, such as bicyclic peptide ligands, having specificity for transferrin receptor 1 (TfR1). The invention also encompasses pharmaceutical compositions comprising said peptide ligands and the use of said peptide ligands and pharmaceutical compositions for the prevention, inhibition or treatment of diseases or conditions by TfR1-mediated delivery of therapeutic agents.

Description

[Field of the Invention]

[0001] The present invention relates to peptide ligands, such as bicyclic peptide ligands, that are specific for transferrin receptor 1 (TfR1). The present invention also includes pharmaceutical compositions comprising the peptide ligands and the use of the peptide ligands and pharmaceutical compositions for preventing, inhibiting or treating diseases or disorders through TfR1-mediated delivery of therapeutic agents. [Background of the invention]

[0003] Cyclic peptides are able to bind to protein targets with high affinity and specificity and are therefore an attractive class of molecules for the development of therapeutic agents. In fact, several cyclic peptides have been successfully used in clinical practice, such as the antimicrobial peptide vancomycin, the immunosuppressive drug cyclosporin or the anticancer drug octreotide (Driggers et al. (2008), Nat. Rev. Drug. Discov. 7 (7), 608-24). The good binding performance is due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an arginine-glycine-aspartate motif that binds to integrin αVb3 (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 combined with urokinase-type plasminogen activator ( Zhao et al. (2007), J. Struct. Biol. 160(1), 1-10).

[0004] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss when binding to a target and resulting in higher binding affinity. Reduced flexibility can also result in locking of target-specific conformations, improving binding specificity compared to linear peptides. This effect has been demonstrated by an effective selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity for other MMPs when its ring is open (Cherney et al. (1998), J. Med. Chem. 41 (11), 1749-51). The favorable binding properties achieved by macrocyclization are more evident in polycyclic peptides with multiple peptide rings, such as in vancomycin, nisin, and actinomycin.

[0005] Different research groups have previously tethered peptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen et al. used tri(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings onto synthetic scaffolds to achieve structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for generating candidate drug compounds, wherein the compounds are generated by attaching cysteine-containing peptides to molecular scaffolds 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).

[0006] Combinatorial methods based on phage display have been developed to generate and screen large bicyclic peptide libraries against target targets (Heinis et al. (2009), Nat. Chem. Biol. 5(7), 502-7 and WO 2009 / 098450). Briefly, two regions containing six random amino acids and three cysteine ​​residues (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) were displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule scaffold. [Summary of the invention]

[0007] According to a first aspect of the present invention, there is provided a peptide ligand specific for transferrin receptor 1 (TfR1), comprising an amino acid sequence selected from the group consisting of:

[0008] C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, also referred to herein as BCY23180);

[0009] C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, also referred to herein as BCY23182);

[0010] CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, also referred to herein as BCY23184);

[0011] CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, also referred to herein as BCY23185);

[0012] CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, also referred to herein as BCY23186);

[0013] CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, also referred to herein as BCY23187);

[0014] CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO:9, also referred to herein as BCY23188);

[0015] CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, also referred to herein as BCY23200);

[0016] CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, also referred to herein as BCY23201);

[0017] CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, also referred to herein as BCY23202);

[0018] CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, also referred to herein as BCY23203);

[0019] CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, also referred to herein as BCY23204);

[0020] CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, also referred to herein as BCY23205);

[0021] CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, also referred to herein as BCY23206);

[0022] CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, also referred to herein as BCY23207);

[0023] CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, also referred to herein as BCY23208);

[0024] CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, also referred to herein as BCY23209);

[0025] CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, also referred to herein as BCY23210);

[0026] CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, also referred to herein as BCY23211);

[0027] CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, also referred to herein as BCY23216);

[0028] CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, also referred to herein as BCY23217);

[0029] CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, also referred to herein as BCY23218);

[0030] CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, also referred to herein as BCY23219);

[0031] CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, also referred to herein as BCY23220);

[0032] CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, also referred to herein as BCY23221);

[0033] CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, also referred to herein as BCY23222);

[0034] CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, also referred to herein as BCY23223);

[0035] CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, also referred to herein as BCY23224);

[0036] CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, also referred to herein as BCY23230);

[0037] CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO:48, also referred to herein as BCY23231); and

[0038] CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, also referred to herein as BCY23515),

[0039] Wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamate, HyP represents hydroxyproline, HSe 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.

[0040] 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 in the peptide ligand are covalently bonded to a molecular scaffold such that two polypeptide rings are formed on the molecular scaffold.

[0041] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or a bicyclic peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0042] According to a further aspect of the present invention, there is provided a peptide ligand, a bicyclic peptide ligand or a pharmaceutical composition as defined herein for use in preventing, inhibiting or treating a disease or disorder by TfR1 mediated delivery of a therapeutic agent. [Detailed description of the invention]

[0044]

Peptide Ligand

[0045] According to a first aspect of the present invention, there is provided a peptide ligand specific for transferrin receptor 1 (TfR1), comprising an amino acid sequence selected from the group consisting of:

[0046] C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, also referred to herein as BCY23180);

[0047] C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, also referred to herein as BCY23182);

[0048] CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, also referred to herein as BCY23184);

[0049] CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, also referred to herein as BCY23185);

[0050] CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, also referred to herein as BCY23186);

[0051] CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, also referred to herein as BCY23187);

[0052] CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO:9, also referred to herein as BCY23188);

[0053] CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, also referred to herein as BCY23200);

[0054] CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, also referred to herein as BCY23201);

[0055] CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, also referred to herein as BCY23202);

[0056] CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, also referred to herein as BCY23203);

[0057] CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, also referred to herein as BCY23204);

[0058] CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, also referred to herein as BCY23205);

[0059] CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, also referred to herein as BCY23206);

[0060] CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, also referred to herein as BCY23207);

[0061] CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, also referred to herein as BCY23208);

[0062] CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, also referred to herein as BCY23209);

[0063] CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, also referred to herein as BCY23210);

[0064] CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, also referred to herein as BCY23211);

[0065] CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, also referred to herein as BCY23216);

[0066] CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, also referred to herein as BCY23217);

[0067] CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, also referred to herein as BCY23218);

[0068] CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, also referred to herein as BCY23219);

[0069] CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, also referred to herein as BCY23220);

[0070] CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, also referred to herein as BCY23221);

[0071] CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, also referred to herein as BCY23222);

[0072] CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, also referred to herein as BCY23223);

[0073] CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, also referred to herein as BCY23224);

[0074] CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, also referred to herein as BCY23230);

[0075] CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO:48, also referred to herein as BCY23231); and

[0076] CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, also referred to herein as BCY23515), or

[0077] a pharmaceutically acceptable salt of the peptide ligand,

[0078] Wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamate, HyP represents hydroxyproline, HSe 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.

[0079] In a further aspect of the present invention, a peptide ligand specific for transferrin receptor 1 (TfR1) comprises an amino acid sequence selected from the group consisting of:

[0080] C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 1, also referred to herein as BCY23180);

[0081] C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 2, also referred to herein as BCY23181);

[0082] C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 3, also referred to herein as BCY23182);

[0083] CP[Oxa]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 4, also referred to herein as BCY23183);

[0084] CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 5, also referred to herein as BCY23184);

[0085] CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 6, also referred to herein as BCY23185);

[0086] CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 7, also referred to herein as BCY23186);

[0087] CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 8, also referred to herein as BCY23187);

[0088] CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO:9, also referred to herein as BCY23188);

[0089] CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (SEQ ID NO: 10, also referred to herein as BCY23189);

[0090] CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (SEQ ID NO: 11, also referred to herein as BCY23190);

[0091] CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (SEQ ID NO: 12, also referred to herein as BCY23191);

[0092] CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (SEQ ID NO: 13, also referred to herein as BCY23192);

[0093] CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (SEQ ID NO: 14, also referred to herein as BCY23193);

[0094] CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (SEQ ID NO: 15, also referred to herein as BCY23194);

[0095] CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (SEQ ID NO: 16, also referred to herein as BCY23195);

[0096] CP[HyP]DAYLSC[tBuGly]SYCEPW (SEQ ID NO: 17, also referred to herein as BCY23196);

[0097] CP[HyP]DAYLDC[tBuGly]SYCEPW (SEQ ID NO: 18, also referred to herein as BCY23197);

[0098] CP[HyP]DAYLYC[tBuGly]SYCEPW (SEQ ID NO: 19, also referred to herein as BCY23198);

[0099] CP[HyP]DAYLNC[tBuGly]SYCEPW (SEQ ID NO: 20, also referred to herein as BCY23199);

[0100] CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 21, also referred to herein as BCY23200);

[0101] CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 22, also referred to herein as BCY23201);

[0102] CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 23, also referred to herein as BCY23202);

[0103] CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 24, also referred to herein as BCY23203);

[0104] CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 25, also referred to herein as BCY23204);

[0105] CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 26, also referred to herein as BCY23205);

[0106] CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 27, also referred to herein as BCY23206);

[0107] CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 28, also referred to herein as BCY23207);

[0108] CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 29, also referred to herein as BCY23208);

[0109] CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 30, also referred to herein as BCY23209);

[0110] CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 31, also referred to herein as BCY23210);

[0111] CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 32, also referred to herein as BCY23211);

[0112] CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 33, also referred to herein as BCY23216);

[0113] CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 34, also referred to herein as BCY23217);

[0114] CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 35, also referred to herein as BCY23218);

[0115] CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 36, also referred to herein as BCY23219);

[0116] CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 37, also referred to herein as BCY23220);

[0117] CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 38, also referred to herein as BCY23221);

[0118] CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 39, also referred to herein as BCY23222);

[0119] CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 40, also referred to herein as BCY23223);

[0120] CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 41, also referred to herein as BCY23224);

[0121] CP[HyP]DAYLGC[tBuGly]TYCEPW (SEQ ID NO: 42, also referred to herein as BCY23225);

[0122] CP[HyP]DAYLGC[tBuGly]DYCEPW (SEQ ID NO: 43, also referred to herein as BCY23226);

[0123] CP[HyP]DAYLGC[tBuGly]EYCEPW (SEQ ID NO: 44, also referred to herein as BCY23227);

[0124] CP[HyP]DAYLGC[tBuGly]NYCEPW (SEQ ID NO: 45, also referred to herein as BCY23228);

[0125] CP[HyP]DAYLGC[tBuGly]QYCEPW (SEQ ID NO: 46, also referred to herein as BCY23229);

[0126] CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 47, also referred to herein as BCY23230);

[0127] CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 48, also referred to herein as BCY23231);

[0128] CP[HyP]DAYLGCYSYCEPW (SEQ ID NO: 49, also referred to herein as BCY23514); and

[0129] CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 50, also referred to herein as BCY23515),

[0130] Wherein Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxy-phenylalanine, Gla represents L-γ-carboxyglutamate, HyP represents hydroxyproline, HSe 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.

[0131] It is understood that each peptide ligand of the present invention comprises an N-terminal acetyl group and a C-terminal CONH 2 base.

[0132] It will also be appreciated that the term "specific for TfR1" refers to the ability of a peptide ligand to bind to transferrin receptor 1 (TfR1). It will also be appreciated that the differential effects of a peptide ligand on TfR1 depend on the precise epitope bound. For example, the effect will be inhibitory (i.e., the peptide ligand hinders / inhibits the binding of transferrin to TfR1) or non-inhibitory (i.e., the peptide ligand does not hinder / inhibit the binding of transferrin to TfR1).

[0133] In a further embodiment, the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium or ammonium salt.

[0134]

Bicyclic peptide ligand

[0135] 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 in the peptide ligand are covalently bonded to a molecular scaffold such that two polypeptide rings are formed on the molecular scaffold.

[0136] In one embodiment, the molecular scaffold is a derivative of TATB having the following structure:

[0137]

[0138] where * indicates the attachment points of three cysteine ​​residues.

[0139] For the purpose of this description, it is assumed that the bicyclic peptide is cyclized with TATB to produce a trisubstituted structure. However, it is clear from the description of the invention presented herein that the cyclization can be performed with any suitable molecular scaffold that forms a covalent bond with the reactive groups of the polypeptide to form at least two polypeptide rings. The cyclization occurs at the first, second and third cysteine ​​residues, respectively.

[0140] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry). Standard techniques are used for molecular biology, genetics, and biochemical methods (see 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 ... th ed., John Wiley & Sons, Inc.), which is incorporated herein by reference.

[0141]

serial number

[0142] When referring to the amino acid residue positions within the peptides of the present invention, cysteine ​​residues are omitted from the numbering because they are invariant, and thus, the numbering of the amino acid residues within the peptides of the present invention is as follows:

[0143] 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).

[0144]

Molecule format

[0145] N-terminal or C-terminal extensions of the bicyclic core sequence were added to the left or right side of the sequence, separated by a hyphen. For example, N-terminal biotin-G-Sar 5 The tail is represented as:

[0146] [Biot]-G-[SAR 5 ]-A-(SEQ ID NO: X).

[0147]

Reverse peptide sequence

[0148] In view of the disclosure in Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is envisioned that the peptide sequences disclosed herein will also find use in their retro-inverso form, i.e., the sequences are reversed (i.e., the N-terminus becomes the C-terminus and vice versa) and their stereochemistry is also reversed (i.e., the D-amino acids become the L-amino acids and vice versa).

[0149] [Peptide ligand definition]

[0150] Peptide ligands, as referred to herein, refer to peptides, peptides or peptidomimetics that are covalently bound to a molecular scaffold. Typically, these peptides, peptides or peptidomimetics include peptides with natural or non-natural amino acids, two or more reactive groups (i.e., cysteine ​​residues) that are capable of forming a covalent bond with the scaffold, and a sequence located between the reactive groups, which is called a loop sequence because it forms a loop when the peptide, peptide or peptidomimetics is bound to the scaffold. In this case, the peptide, peptide or peptidomimetics contains at least three cysteine ​​residues and forms at least two loops on the scaffold.

[0151]

Advantages of peptide ligands

[0152] Certain bicyclic peptides of the present invention have many advantageous properties that enable them to be considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. These advantageous properties include:

[0153] Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations;

[0154] Protease stability. In most cases, bicyclic peptide ligands should exhibit stability against plasma proteases, epithelial (“membrane-anchored”) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that bicyclic peptide lead candidates can be developed in animal models and administered to humans with confidence;

[0155] The ideal solubility curve. This is a function of the ratio of charged and hydrophilic to hydrophobic residues and intra / inter-molecular hydrogen bonding, which is important for formation and absorption purposes; and

[0156] Optimal plasma half-life in circulation. 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 will depend on the requirement for continuous exposure (to obtain maximum therapeutic effect) versus the requirement for short exposure time (to minimize toxicological effects caused by continuous exposure to the drug).

[0157] [Pharmaceutically acceptable salts]

[0158] It will be appreciated that salt forms are within the scope of the invention and reference to a peptide ligand includes salt forms of the ligand.

[0159] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods, such as 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 an appropriate base or acid in water or an organic solvent, or in a mixture of the two.

[0160] Acid addition salts (mono or di-salts) can be formed with a variety of acids, including inorganic and organic acids. Examples of acid addition salts include mono or di-salts formed with acids selected from 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, butyric acid, (+) camphor, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, sweet acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, creatine enanthate, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, malic acid, succinic ... Uric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), ether sulfide ions, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactose, 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, dihydroxy acid, hydrogen phosphate, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0161] One class of special salts consists of salts formed from acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylic), ethanesulfonic, naphthalenesulfonic, valeric, propionic, butyric, malonic, glucuronic and lactobionic acids. One special salt is the hydrochloride. Another special salt is the acetate.

[0162] If the compound is anionic, or has a functional group that could be anionic (e.g. -COOH could be -COO - ), then the salt can be formed with an organic or inorganic base to produce a suitable cation. 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 + Suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g. NH 3 R+ NH 2 R 2 + 、NHR 3 + NR 4 + Some examples of suitable substituted ammonium ions are derived from the following: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, benzylamine, 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 + .

[0163] When the peptides of the present invention contain amine functional groups, these peptides can form quaternary ammonium salts, for example, by reacting with alkylating agents according to methods well known to the skilled person. Such quaternary ammonium compounds fall within the scope of the peptides of the present invention.

[0164]

Modified derivatives

[0165] It will be appreciated that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacers; substitution of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; substitution of one or more amino acid residues with one or more alternative amino acids, such as 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 the bicyclic peptide ligand; substitution of one or more peptide bonds with alternative bonds; modification of the peptide backbone length; substitution of a hydrogen on the α-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 to functionalize the amino acids; and introduction or substitution of amino acids with orthogonal reactivity suitable for functionalization, such as amino acids containing azide or alkyne groups that allow functionalization with alkyne or azide-containing moieties, respectively.

[0166] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In further embodiments, wherein the modified derivatives include N-terminal modifications using suitable amino-reactive chemistry, and / or C-terminal modifications using suitable carboxylic acid-reactive chemistry. In further embodiments, the N-terminal or C-terminal modifications include the addition of effector groups, including but not limited to cytotoxic agents, radiochelators, or chromophores.

[0167] In a further embodiment, the modified derivative includes an N-terminal modification. In a further embodiment, the N-terminal modification includes an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal residue is blocked by acetic anhydride or other appropriate reagents, resulting in the molecule being acetylated at the N-terminus. This embodiment provides the advantage of removing potential recognition points of aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0168] In an alternative embodiment, the N-terminal modification involves the addition of a molecular spacer that facilitates conjugation of the effector group and retention of the potency of the bicyclic peptide towards its target.

[0169] In a further embodiment, the modified derivative includes a C-terminal modification. In a further embodiment, the C-terminal modification includes an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis, resulting in a C-terminal amidated molecule. This embodiment provides the advantage of removing potential recognition points of carboxypeptidases and reduces the possibility of proteolytic degradation of the bicyclic peptide.

[0170] In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isostructural / isoelectronic side chains can be selected that are neither recognized by degradative proteases nor have any adverse effects on target efficacy.

[0171] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these involve proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib) and cyclic amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0172] In one embodiment, the improved derivative comprises the addition of a spacer. In a further embodiment, the modified derivative comprises the addition of a spacer to the N-terminal cysteine ​​and / or the C-terminal cysteine.

[0173] In one embodiment, the modified derivatives include replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues. In a further embodiment, the modified derivatives include replacing tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the drug stability spectrum of the resulting bicyclic peptide ligands.

[0174] In one embodiment, the modified derivative includes replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative includes replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged amino acid residues and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues can affect the degree of plasma protein binding, thereby affecting the concentration of the free effective part in the plasma, while charged amino acid residues (particularly arginine) may affect the interaction between the peptide and the phospholipid membrane on the cell surface. The combination of the two may affect the half-life, distribution volume and exposure of the peptide drug, and can be customized according to clinical endpoints. In addition, the correct combination and quantity of charged and hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

[0175] In one embodiment, the modified derivative comprises replacing 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 the tendency of D amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS, 102 (2), 413-418).

[0176] In one embodiment, the modified derivatives include removal of any amino acid residues and substitution with alanine, such as D-alanine. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack sites.

[0177] It should be noted that each of the above modifications is used to intentionally improve the potency or stability of the peptide. Further potency improvements based on modification may be achieved through the following mechanisms:

[0178] Incorporation of hydrophobic moieties that exploit the hydrophobic effect and result in lower dissociation rates, leading to higher affinity;

[0179] Incorporation of charged groups that exploit long-range ionic interactions, resulting in faster rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0180] Adding additional constraints to the peptide, for example by properly constraining the side chains of the amino acids so that the loss of entropy upon target binding is minimal, constraining the torsion angles of the backbone so that the loss of entropy upon target binding is minimal, and introducing additional cyclizations in the molecule for the same reasons.

[0181] (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0182] Isotope changes

[0183] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present 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 usually present in nature, as well as peptide ligands of the present invention, in which a metal chelating group is attached (referred to as an "effector") capable of retaining the relevant (radio)isotope, and peptide ligands of the present invention, in which certain functional groups are covalently substituted by relevant (radio)isotopes or isotope-labeled functional groups.

[0184] Examples of isotopes suitable for inclusion in the peptide ligands of the invention include 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 Cl, fluorine, such as 18 F, iodine, such as 123 I, 125 I and 131 I, nitrogen, such as 13 N and 15 N, oxygen, such as 15 Oh, 17 O and 18 O, phosphorus, such as 32 P, sulfur, 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.

[0185] Certain isotopically labeled peptide ligands of the invention, such as those incorporating radioactive isotopes, can be used in drug and / or substrate tissue distribution studies and in clinical assessment of the presence and / or absence of a target on diseased tissue. The peptide ligands of the invention can further have valuable diagnostic properties because they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with a labeling agent, such as a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance (e.g., luminol, luminol derivatives, fluorescein, aequorin and luciferase), etc. The radioactive isotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C, are particularly suitable for this purpose given their ease of incorporation and readily available means of detection.

[0186] Using heavier isotopes (such as deuterium), 2 H(D) substitution may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0187] Using positron emitting isotopes (such as 11 C. 18 F. 15 O and 13 N) substitution, can be used in positron emission tomography (PET) studies to examine target occupancy.

[0188] Isotopically labeled compounds of the 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 labeled reagent in place of the unlabeled reagent previously employed.

[0189]

Molecular Scaffold

[0190] In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. "Non-aromatic molecular scaffold" as referred to herein refers to any molecular scaffold as defined herein that does not contain an aromatic (ie, unsaturated) carbocyclic or heterocyclic ring system.

[0191] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53 (6) 1602-1606.

[0192] As described in the above-mentioned document, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0193] 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.

[0194] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of the polypeptide to form covalent bonds.

[0195] The molecular scaffold may comprise chemical groups that form linkages with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0196] 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 1,3,5-triacryloylhexahydro-s-triazine (TATA)):

[0197]

[0198] Thus, after cyclization with the bicyclic peptide of the invention on three cysteine ​​residues, the molecular scaffold forms a trisubstituted 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure:

[0199]

[0200] where * indicates the attachment points of three cysteine ​​residues.

[0201] In an alternative embodiment, the molecular scaffold is 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine (TATB):

[0202]

[0203] Thus, after cyclization with the bicyclic peptide of the present invention on a cysteine ​​residue, the molecular scaffold forms a trisubstituted 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine derivative of TATB having the following structure:

[0204]

[0205] where * indicates the attachment points of three cysteine ​​residues.

[0206]

synthesis

[0207] The peptides of the present invention can be synthesized by standard techniques and then reacted with molecular scaffolds in vitro. When doing this, standard chemical reagents can be used. This makes it possible to quickly prepare soluble materials on a large scale for further downstream experiments or verification. This method can be completed using conventional chemical methods, such as those disclosed in Timmerman et al. (supra).

[0208] Therefore, the present invention also relates to the manufacture of the polypeptides or conjugates selected herein, wherein the manufacture comprises optional further steps as described below. In one embodiment, these steps are performed on the final product polypeptide / conjugate made by chemical synthesis.

[0209] In making a conjugate or complex, amino acid residues in the target polypeptide may be selectively substituted.

[0210] The peptide may also be extended, for example by incorporating a further loop, thereby introducing multiple specificities.

[0211] To extend the peptide, it can be simply chemically extended using orthogonally protected lysine (and analogs) at its N-terminus or C-terminus or within a ring using standard solid or liquid phase chemistry. Standard (bio) coupling techniques can be used to introduce an activated or activatable N-terminus or C-terminus. Alternatively, it can be added by fragment condensation or native chemical ligation, such as described in (Dawson et al., 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779), or by enzymes, such as using subpeptidases such as (Chang et al., Proc Natl Acad Sci USA. 1994 December 20; 91 (26): 12544-8 or in Hikari et al., Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0212] Alternatively, the peptides may be extended or modified by further coupling via disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA or TATB) may be added during the chemical synthesis of the first peptide so as to react with the three cysteine ​​groups; a further cysteine ​​or thiol may then be appended to the N- or C-terminus of the first peptide so that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0213] Furthermore, the addition of other functional groups or effector groups can be accomplished in the same manner, using appropriate chemistry, either at the N-terminus or C-terminus or via side chain coupling. In one embodiment, the coupling is performed in such a way that it does not prevent the activity of either entity.

[0214] [Drug composition]

[0215] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0216] Typically, the peptide ligands present will be used in purified form with a pharmacologically appropriate excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, and lactated Ringer's solution. If it is necessary to keep the polypeptide complex in suspension, a physiologically acceptable suitable adjuvant can be selected from thickeners such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, and alginate.

[0217] 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).

[0218] The peptide ligands of the invention may be used as compositions for administration alone or in combination with other agents. These may include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Further examples of other agents that may be administered alone or in combination with the peptide ligands of the invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic and antithrombotic factors. Pharmaceutical compositions may include a "mixture" of various cytotoxic or other agents with the protein ligands of the invention, or even a combination of polypeptides with different specificities selected according to the invention, such as polypeptides selected to use different target ligands, whether or not they are mixed prior to administration.

[0219] The route of administration of the pharmaceutical composition according to the present invention can be any of those commonly known to those of ordinary skill in the art. For treatment, the peptide ligands of the present invention can be applied to any patient according to standard techniques. Administration can be carried out by any appropriate means, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, by pulmonary approach, or suitably by direct infusion of a catheter. Preferably, the pharmaceutical composition according to the present invention will be administered intravenously. The dosage and frequency of administration will depend on the patient's age, sex and condition, concurrent administration of other drugs, contraindications and other parameters that the clinician will consider.

[0220] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been proven to be effective and lyophilization and reconstitution techniques known in the art can be used. Those skilled in the art will recognize that lyophilization and reconstitution will result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[0221] Compositions containing existing peptide ligands or mixtures thereof can be used for prevention and / or treatment. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, regulation, killing or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease, but is typically within the range of 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with a more commonly used dose of 0.05 to 2.0 mg / kg / dose. For preventive applications, compositions containing current peptide ligands or mixtures thereof can also be administered in similar or slightly lower doses.

[0222] Compositions containing peptide ligands according to the present invention can be used in preventive and therapeutic settings to help alter, inactivate, kill or remove selected target cell populations in mammals. In addition, the peptide ligands described herein can be used to selectively kill, deplete or otherwise effectively remove target cell populations from heterogeneous cell collections in vitro or in vitro. Blood from a mammal can be combined with a selected peptide ligand in vitro to kill unwanted cells or otherwise remove them from the blood so that they can be returned to the mammal according to standard techniques.

[0223]

Therapeutic Uses

[0224] The bicyclic peptides of the invention have particular use 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, inhibiting or treating a disease or condition by TfR1 mediated delivery of a therapeutic agent.

[0225] Transferrin is a glycoprotein found in vertebrates that binds iron (Fe) and thus mediates the transport of iron (Fe) through plasma. It is produced in the liver and contains two Fe3+ The binding site of the atom. Human transferrin is encoded by the TF gene and is produced as a 76 kDa glycoprotein.

[0226] The transferrin glycoprotein binds iron tightly but reversibly. Although less than 0.1% (4 mg) of total body iron is bound to transferrin, it forms the most important iron pool with the highest turnover rate (25 mg / 24 h). Transferrin has a molecular weight of approximately 80 kDa and contains two specific high-affinity Fe(III) binding sites. Transferrin has an extremely high affinity for Fe(III) (association constant 10 at pH 7.4). 20 M -1 ), but gradually decreases as the pH decreases below neutral. Transferrin is not limited to binding iron, but also binds to different metal ions. These glycoproteins are located in various body fluids of vertebrates.

[0227] When not bound to iron, transferrin is called "apotransferrin."

[0228] In one embodiment, the transferrin is a mammalian transferrin. In a further embodiment, the mammalian transferrin is a human transferrin. In one embodiment, the human transferrin is a human transferrin receptor 1 (TfR1; also known as CD71).

[0229] TfR1 binding peptides may be useful in treating neurological diseases. Examples of such neurological diseases include, but are not limited to:

[0230] Neuropathic diseases, neurodegenerative diseases, cancer, eye diseases, epilepsy, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders and central nervous system inflammation.

[0231] In one embodiment, the neurological disease is in a human subject. It is understood that the dosage and / or dosing frequency is adjusted to reduce the concentration of the peptide ligand to which the erythrocytes are exposed. In a further embodiment, the treatment further comprises the step of monitoring the human subject for erythrocyte depletion.

[0232] The term "prevention" as used herein refers to administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after the induction event but prior to clinical manifestation of the disease. "Treatment" includes administration of the protective composition after disease symptoms become apparent.

[0233] Animal model systems are available for screening peptide ligands for effectiveness in preventing or treating disease. The present invention facilitates the use of animal model systems, which allows the development of polypeptide ligands that can cross-react with human and animal targets to allow the use of animal models.

[0234] Transferrin receptor 1 (TfR1) is an extensively studied model receptor-ligand system that has provided considerable insights into the cellular properties and mechanisms of internalization and endocytic sorting of nutrient / scavenger receptor cargo (Qian et al (2002) Pharmacological Reviews 54(4), 561-587). TfR1 is known to undergo constitutive endocytosis and recycle to the plasma membrane and has pH-dependent ligand binding to properly sort endocytic cargo. Anti-TfR1 antibodies were previously considered the mainstay of targeting TfR1 in oligonucleotide therapy, however, the current Tfr1-binding peptide ligands of the present invention have the potential to demonstrate effective and complete knockdown of gene expression in skeletal and cardiac muscle via systemically delivered TfR1-bicyclic peptide-siRNA conjugates.

[0235] Therefore, in view of this mechanism, it is believed that the peptide ligands of the present invention can be used as tissue delivery complexes, for example, to deliver Tfr1-peptide ligand-payload (ie, siRNA) complexes to tissue cells, particularly muscle cells.

[0236] Therefore, according to a further aspect of the present invention, there is provided a tissue delivery complex comprising a peptide ligand of the present invention that binds to TfR1 in combination with a payload (eg another peptide, a small molecule drug or an oligonucleotide, particularly siRNA).

[0237] Therefore, the tissue delivery complex can be used to treat musculoskeletal diseases. Examples of suitable musculoskeletal diseases include, but are not limited to:

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] The present invention will be further described below in conjunction with the following examples. [Example]

[0248]

Materials and methods

[0249]

Preparation of Bicyclic Peptide Ligands (General Method)

[0250] Using standard Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis, bicyclic peptides are synthesized on Rink amide resin by manual coupling (for large scale) or using Biotage SyroII automatic peptide synthesizer (for small scale). After TFA-based cleavage from the resin, the peptide is precipitated with ether and dissolved in 50:50 acetonitrile / water. Then use ammonium bicarbonate (100mM) as a base and use 1.3 times the amount of scaffold cyclization crude peptide (~1mM concentration). Cyclization is completed by matrix-assisted laser desorption ionization flight time (MALDI-TOF) or LC-MS. After completion, the cyclization reaction is quenched using N-acetylcysteine ​​(relative to 10 times the amount of peptide), and the solution is freeze-dried. The residue is dissolved in an appropriate solvent and purified by RP-HPLC. Merge peptide components with sufficient purity and correct molecular weight (verified by MALDI-TOF and HPLC or LC-MS) and freeze-dried. Concentration was determined by UV absorption using the extinction coefficient at 280 nm, which is based on the tryptophan / tyrosine content.

[0251] Unless otherwise stated, all amino acids were used in the L configuration.

[0252]

Biological Data

[0253] The bicyclic peptide ligands of the invention were tested in the following assays:

[0254] 【1.TfR1 SPR binding assay】

[0255] Biacore experiments can be performed to determine the k of various peptides binding to TfR1. a (M -1 s -1 ), k d (s -1 ), K D (nM) value.

[0256] Recombinant human and cynomolgus monkey TfR1 were obtained from the bicyclic 6 Tagged TfR1 (aa89-760) (ACRO Biosystems, CD1-H5243 and TFR-C524a).

[0257] To analyze TfR1 peptide binding, Cytiva NTA chips were run using the capture / coupling method using a Biacore T200 or S200 instrument at 25°C with 25 mM HEPES, 0.1 M NaCl, 0.05% Tween 20 pH 7.4 as the running buffer. Fixation was performed as follows. The chip was pre-equilibrated by injecting 500 mM EDTA (pH 8) and then eluted with 5 mM NiSO 4 Activation. The surface was then activated using standard amine coupling chemistry. Briefly, the carboxymethyl dextran surface was activated with 0.4M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1M N-hydroxysuccinimide (NHS) in a 1:1 ratio. TfR1 protein (human or cynomolgus monkey) was then captured onto the activated surface after dilution to 200nM and 250nM in electrophoresis buffer, respectively. The residual activated group was blocked by injection of 1M ethanolamine (pH8.5):HBS-N (1:1) for 7 minutes. The reference surface was activated and blocked as described above, without TfR1 protein capture. Capture levels ranged from 1,500 to 5,000 RU, depending on the individual study buffer was changed to 25mM HEPES, 0.1M NaCl, 0.05% Tween 20, pH 7.4, 1% DMSO.

[0258] A dilution series of the test peptides was prepared in this buffer, with the highest peptide concentration being 5 μM, followed by six 2-fold dilutions. SPR analysis was performed at 25°C at a flow rate of 30 μl / min, with an association time of 160 s and a dissociation time of 700-800 s. Data were corrected for DMSO to exclude volume effects. All data were double-checked for blank injections and control surfaces using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software version 2.0c (BioLogic Software). The data were fit using a simple 1:1 binding model, allowing for bulk transport effects where appropriate.

[0259] [Table 1: Human TfR1 SPR binding assay results]

[0260] BCY number SPR human TfR1 KD (nM) BCY23180 50 BCY23182 22 BCY23184 13 BCY23185 12 BCY23186 27 BCY23187 38 BCY23188 109 BCY23200 9.5 BCY23201 17 BCY23202 23 BCY23203 283 BCY23204 9 BCY23205 11 BCY23206 10 BCY23207 7.7 BCY23208 6 BCY23209 5.6 BCY23210 8.6 BCY23211 7.7 BCY23216 158 BCY23217 117 BCY23218 55 BCY23219 51 BCY23220 72 BCY23221 120 BCY23222 58 BCY23223 50 BCY23224 1140 BCY23230 15 BCY23231 25 BCY23515 85

Claims

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

2. The peptide ligand according to claim 1, comprising an N-terminal acetyl group and a C-terminal CONH 2 base.

3. The peptide ligand according to claim 1 or 2, in, Pharmaceutically acceptable salts are selected from the free acid or sodium, potassium, calcium or ammonium salts.

4. A bicyclic peptide ligand comprising a peptide ligand according to any one of claims 1 to 3, in, The first, second and third cysteine ​​residues within the peptide ligand are covalently bonded to the molecular scaffold, so that two polypeptide loops are formed on the molecular scaffold.

5. The bicyclic peptide ligand according to claim 4, in, The molecular scaffold is a derivative of TATB, which has the following structure: where * indicates the attachment points of three cysteine ​​residues.

6. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 3 or a bicyclic peptide ligand according to claim 4 or 5, in combination with one or more pharmaceutically acceptable excipients.

7. A peptide ligand according to any one of claims 1 to 3, or a bicyclic peptide ligand according to claim 4 or 5, or a pharmaceutical composition according to claim 6, for use in preventing, inhibiting or treating a disease or condition by TfR1-mediated delivery of a therapeutic agent.

8. A tissue delivery complex comprising a peptide ligand according to any one of claims 1 to 3 or a bicyclic peptide ligand according to claim 4 or 5, bound to Tfr1 with a payload (such as an oligonucleotide, in particular siRNA).

9. The tissue delivery complex according to claim 8, which is a muscle tissue delivery complex.

10. The tissue delivery complex according to claim 8 or 9, for use in treating musculoskeletal diseases.

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