Bicyclic peptide ligands specific for binding tin-4

Abstract

The present invention relates to bicyclic peptide ligands specific for binding tin-4. In particular, the present invention relates to polypeptides covalently bound to molecular scaffolds, such that two or more peptide rings oppose each other between the attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of binding tin-4. The invention also encompasses drug conjugates comprising said peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising said peptide ligands and drug conjugates, and the use of said peptide ligands and drug conjugates in the prevention, inhibition or treatment of binding group-4 mediated diseases or conditions.

Description

[0001] This application is a divisional application of Chinese patent application No. 2019800548178 “Bicyclic peptide ligands specific for bindin-4” filed on June 21, 2019. Technical Field

[0002] The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide rings subtend between attachment points of the scaffold. In particular, the present invention describes peptides that are high-affinity binders to integrin-4. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and uses of the peptide ligands and drug conjugates in preventing, inhibiting, or treating diseases or conditions mediated by integrin-4. Background Art

[0003] Cyclic peptides are able to bind to protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutics. In fact, several cyclic peptides have been successfully used clinically, such as the antimicrobial peptide vancomycin, the immunosuppressant drug cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The good binding properties are derived from 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 hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), binds to integrin αVb3 cyclic peptides with an Arg-Gly-Asp motif (Xiong et al. (2002), Science 296(5565), 151-5), or upain-1 ( 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 a smaller loss of entropy when binding to the target and resulting in higher binding affinity. The reduced flexibility also results in locking the target-specific conformation, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent, selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity for other MMPs when its ring is opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties obtained by macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring (e.g., in vancomycin, nisin, and actinomycin).

[0005] Various research groups have previously linked peptides containing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues have used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for generating candidate drug compounds are described in which the compounds are generated by linking cysteine-containing peptides to molecular scaffolds such as TATA (1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one) (Heinis et al. Angew Chem, Int Ed. 2014;53:1602-1606).

[0006] A combinatorial approach based on phage display has been developed to generate and screen large libraries of bicyclic peptides against target targets (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO2009 / 098450). Briefly, a combinatorial library of linear peptides containing two regions of three cysteine ​​residues and six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) is displayed on phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. Summary of the Invention

[0007] According to the first aspect of the present invention, a peptide ligand specific for nectin-4 is provided, which comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two ring sequences, and the molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold.

[0008] According to another aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0009] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0010] According to another aspect of the present invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, inhibiting or treating a disease or condition mediated by Integrin-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Where present in the figures, error bars represent the standard error of the mean (SEM).

[0012] Figures 1 to 7 : Tumor volume trajectories after administration of BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, and BCY8255 to female BALB / c nude mice bearing NCI-H292 xenografts.

[0013] Figures 8 to 10 : Tumor volume trajectory after administration of BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing NCI-H292 xenografts.

[0014] Figures 11 to 15 : Tumor volume trajectory after administration of BCY7825, BCY8245, BCY8253, BCY8254, and BCY8255 to female CB17-SCID mice bearing HT-1376 xenografts.

[0015] Figures 16 to 18 : Tumor volume trajectory after administration of BCY8245, BCY8253, and BCY8255 to female CB17-SCID mice bearing HT-1376 xenografts.

[0016] Figures 19 to 21 : Tumor volume trajectories after administration of BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing Panc2.13 xenografts.

[0017] Figures 22 to 24 : Tumor volume trajectory after administration of BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0018] Figures 25 to 28: Tumor volume trajectory after administration of BCY8549, BCY8550, BCY8783, and BCY8784 (BCY8245 as a control) to female BALB / c nude mice bearing NCI-H292 xenografts.

[0019] Figure 29 : Gating strategy for integrin-4 in mammary gland (T-47D and MDA-MB-468).

[0020] Figure 30 : Gating strategy for nectin-4 in NCI-H292 and NCI-H322.

[0021] Figures 31 to 32 : Gating strategies for Integrin-4 in NCI-H526 and HT1080, respectively.

[0022] Figures 33 to 37 :In bladder cancer (HT1376; Figure 33 ), breast cancer (MDA-MB-468; Figure 34 ), colorectal cancer (HT-29; Figure 35A and HCT-116; Figure 35B ), lung cancer (A549; Figure 36A , NCI-H292; Figure 36B , NCI-H358; Figure 36C and NCI-526; Figure 36D ), and pancreatic cancer (Panc02.13; Figure 37 ) in the gating strategy of nephrin-4.

[0023] Figures 38 to 41 : Tumor volume trajectory after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing A549 xenografts.

[0024] Figures 42 to 45 : Tumor volume trajectory after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing HCT116 xenografts.

[0025] Figures 46 to 49 : Tumor volume trajectory after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female CB17-SCID mice bearing HT-1376 xenografts.

[0026] Figures 50 to 53: Tumor volume trajectory after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0027] Figures 54 to 57 : Tumor volume trajectory after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing NCI-H292 xenografts.

[0028] Figures 58 to 59 : Tumor volume trajectory after administration of BCY8245 and BCY8255 to female Balb / c nude mice bearing NCI-H526 xenografts.

[0029] Figures 60 to 63 : Tumor volume trajectories after administration of BCY8242, BCY8245, BCY8253, and BCY8255 to female Balb / c nude mice bearing Panc2.13 xenografts.

[0030] Figure 64 : Tumor volume trajectory following administration of BCY8245, BCY8781, or a combination of BCY8245 and BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0031] Figure 65 : Tumor volume trajectory following administration of BCY8245 alone or in combination with BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0032] Figures 66 to 71 : Tumor volume trajectories in Lu-01-0412, LU-01-0007, CTG-1771, CTG-1171, CTG-1106, and CTG-0896 PDX xenografts.

[0033] Figure 72 BT8009 (BCY8245) efficacy correlates with expression in CDX / PDX xenografts. Xenografts with low or no integrin-4 expression showed reduced tumor growth rates. Xenografts expressing integrin-4 showed tumor regression. This analysis included both PDX and CDX models, collating values ​​from various reports.

[0034] Figure 73 : MDA-MB-468 cells express integrin-4 and show prolonged retention of MMAE in tumors.

[0035] Figure 74: HCS-data analysis on MDA-MB-468 cell line. DETAILED DESCRIPTION

[0036] In one embodiment, the loop sequence comprises 3, 6, 7, 8 or 9 amino acids. In another embodiment, the loop sequence comprises 3, 6, 7 or 9 amino acids. In yet another embodiment, the loop sequence comprises 3 or 9 amino acids.

[0037] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 3 amino acids and the other of which consists of 9 amino acids.

[0038] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 3 amino acids and the other of which consists of 8 amino acids.

[0039] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 7 amino acids and the other of which consists of 3 amino acids.

[0040] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, both loop sequences consisting of 6 amino acids.

[0041] In one embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0042] C i -P / A / Hyp-F / YG / AC ii -X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii (SEQ ID NO: 38);

[0043] C i -W / APLD / SS / DYWC ii -X5-RIC iii (SEQ ID NO: 39);

[0044] C i -VTTSYDC ii -F / WL / VH / R / TLL / GG / Q / HC iii (SEQ ID NO: 40);

[0045] C i -X6-X7-X8-C ii-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii (SEQ ID NO: 41); and

[0046] C i -W / A / YP / ALD / S / AS / D / P / AYW / 1-Nal-C ii -X s -R / HArg / AIC iii (SEQ ID NO: 42);

[0047] in:

[0048] X1-X5 represent any amino acid residue, including modified and unnatural amino acids;

[0049] X6 represents: Gly; Pro or a non-natural derivative of Pro selected from azetidine (Aze), hydroxyproline (HyP), 4-amino-proline (Pro(4NH)), oxazolidine-4-carboxylic acid (Oxa), octahydroindolecarboxylic acid (Oic) or 4,4-difluoroproline (4,4-DFP); Ala or a non-natural derivative of Ala selected from aminoisobutyric acid (Aib); or sarcosine (Sar);

[0050] X7 represents: Phe or a non-natural derivative of Phe selected from 3-methyl-phenylalanine (3MePhe), 4-methyl-phenylalanine (4MePhe), homophenylalanine (HPhe), 4,4-biphenylalanine (4,4-BPA) or 3,4-dihydroxy-phenylalanine (DOPA); Tyr; or Ala or a non-natural derivative of Ala selected from 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal) or 2-pyridylalanine (2Pal);

[0051] X8 represents: Gly; Ala; Asp; Lys or a non-natural derivative of Lys selected from acetylated lysine (KAc or Lys(Ac)); Phe; Glu; Gln; Leu; Ser; Arg; or cysteic acid (Cya);

[0052] X9 is absent or represents: Met or a non-natural derivative of Met selected from methionine sulfone (Met(O2)); Gln or a non-natural derivative of Gln selected from homoglutamine (HGln); Leu or a non-natural derivative of Leu selected from homoleucine (HLeu) or norleucine (Nle); Lys; Ile; tert-butylalanine (tBuAla); or methylhomoserine (HSe(Me));

[0053] X 10 Representative: Pro; Lys or a non-natural derivative of Lys, which is selected from acetylated lysine (KAc or Lys(Ac)); Arg or a non-natural derivative of Arg, which is selected from 2-amino-4-guanidinobutyric acid (Agb), homoarginine (HArg) or N-methyl-homoarginine; Glu; Ser; Asp; Gln; Ala; hydroxyproline (HyP); or cysteic acid (Cya);

[0054] X 11 represents: Asn or a non-natural derivative of Asn selected from N-methyl-asparagine; Thr; Asp; Gly; Ser; His; Ala or a non-natural derivative of Ala selected from thian-alanine (Thi), 2-(1,2,4-triazol-1-yl)-alanine (1,2,4-TriAz) or β-(4-thiazolyl)-alanine (4ThiAz); Lys; or cysteic acid (Cya);

[0055] X 12 Representative: Trp or a non-natural derivative of Trp, which is selected from azatryptophan (AzaTrp), 5-fluoro-L-tryptophan (5FTrp) or methyltryptophan (TrpMe); or Ala or a non-natural derivative of Ala, which is selected from 1-naphthylalanine (1-Nal) or 2-naphthylalanine (2-Nal);

[0056] X 13 represents: Ser or a non-natural derivative of Ser selected from homoserine (HSer); Ala; Asp; or Thr;

[0057] X 14Representative: Trp or a non-natural derivative of Trp selected from azatryptophan (AzaTrp); Ser; Ala or a non-natural derivative of Ala selected from 2-(1,2,4-triazol-1-yl)-alanine (1,2,4-TriAz), 1-naphthylalanine (1-Nal) or 2-naphthylalanine (2-Nal); Asp; Phe or a non-natural derivative of Phe selected from 3,4-dihydroxy-phenylalanine (DOPA); Tyr; Thr or a non-natural derivative of Thr selected from N-methyl-threonine; tetrahydropyran-4-propionic acid (THP(O)); or dioxo-4-tetrahydrothiopyranylacetic acid (THP(SO2));

[0058] X 15 represents Pro or a non-natural derivative of Pro selected from azetidine (Aze), pipecolic acid (Pip) or oxazolidine-4-carboxylic acid (Oxa);

[0059] X 16 Representative: Ile or a non-natural derivative of Ile selected from N-methyl-isoleucine (NMeIle); Ala or a non-natural derivative of Ala selected from 3-cyclohexyl-alanine (Cha) or cyclopropyl-alanine (Cpa); Pro or a non-natural derivative of Pro selected from hydroxyproline (HyP); Asp; Lys; cyclopentyl-glycine (C5A); tetrahydropyran-4-propionic acid (THP(O)); or dioxo-4-tetrahydrothiopyranyl acetic acid (THP(SO2));

[0060] X 17 represents: Trp or a non-natural derivative of Trp selected from azatryptophan (AzaTrp) or 5-fluoro-L-tryptophan (5FTrp); Phe; Tyr; 1-naphthylalanine (1-Nal); or 2-naphthylalanine (2-Nal);

[0061] Hyp stands for hydroxyproline, 1-Nal stands for 1-naphthylalanine, 2-Nal stands for 2-naphthylalanine, HArg stands for homoarginine and C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0062] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0063] C i -X6-X7-X8-C ii-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii (SEQ ID NO:41)

[0064] Among them X6 to X 17 As defined herein.

[0065] In one embodiment, X6 represents: Pro or a non-natural derivative of Pro selected from azetidine (Aze), hydroxyproline (HyP), 4-amino-proline (Pro(4NH)), oxazolidine-4-carboxylic acid (Oxa), octahydroindolecarboxylic acid (Oic), or 4,4-difluoroproline (4,4-DFP). In another embodiment, X6 represents Pro.

[0066] In one embodiment, X7 represents: Phe or a non-natural derivative of Phe selected from 3-methyl-phenylalanine (3MePhe), 4-methyl-phenylalanine (4MePhe), homophenylalanine (HPhe), 4,4-biphenylalanine (4,4-BPA), or 3,4-dihydroxy-phenylalanine (DOPA); Ala or a non-natural derivative of Ala selected from 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), or 2-pyridylalanine (2Pal). In another embodiment, X7 represents Phe or 1-naphthylalanine (1-Nal). In yet another embodiment, X7 represents 1-naphthylalanine (1-Nal).

[0067] In one embodiment, X8 represents Asp, Arg, Lys or cysteic acid (Cya). In another embodiment, X8 represents D-Asp, D-Arg, D-Lys or D-Cya. In another embodiment, X8 represents D-Asp.

[0068] In one embodiment, X9 represents: Met or a non-natural derivative of Met selected from methionine sulfone (Met(O2)); or Leu or a non-natural derivative of Leu selected from homoleucine (HLeu) or norleucine (Nle). In another embodiment, X9 represents Met or Leu. In another embodiment, X9 represents Met.

[0069] In one embodiment, X 10represents Arg or a non-natural derivative of Arg selected from 2-amino-4-guanidinobutyric acid (Agb), homoarginine (HArg) or N-methyl-homoarginine; or cysteic acid (Cya). In another embodiment, X 10 In another embodiment, X 10 In another embodiment, X 10 Stands for lysine.

[0070] In one embodiment, X 11 represents: Asn or a non-natural derivative of Asn selected from N-methyl-asparagine; Asp; or His; or Cysteic Acid (Cya). In another embodiment, X 11 In another embodiment, X 11 Stands for Asp.

[0071] In one embodiment, X 12 represents: Trp or a non-natural derivative of Trp selected from azatryptophan (AzaTrp), 5-fluoro-L-tryptophan (5FTrp) or methyl-tryptophan (TrpMe). 12 Represents Trp.

[0072] In another embodiment, X 13 represents Ser or a non-natural derivative of Ser selected from homoserine (HSer). In another embodiment, X 13 Stands for Ser.

[0073] In one embodiment, X 14 In another embodiment, X 14 Stands for Thr.

[0074] In one embodiment, X 15 Stands for Pro.

[0075] In one embodiment, X 16 represents: Ile or a non-natural derivative of Ile selected from N-methyl-isoleucine (NMeIle); or Pro or a non-natural derivative of Pro selected from hydroxyproline (HyP). In another embodiment, X 16 Represents: Ile; Pro or a non-natural derivative of Pro selected from hydroxyproline (HyP). In another embodiment, X 16In another embodiment, X 16 Stands for hydroxyproline (HyP).

[0076] In one embodiment, X 17 represents Trp or a non-natural derivative of Trp selected from azatryptophan (AzaTrp) or 5-fluoro-L-tryptophan (5FTrp). 17 Represents Trp.

[0077] In yet another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0078] C i -P-X7-X8-C ii -X9-HArg / Lys-X 11 -WSTPX 16 -WC iii (SEQ ID NO: 213);

[0079] Among them, X7, X8, X9, X 11 and X 16 As defined herein.

[0080] In yet another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0081] C i -P-X7-X8-C ii -X9-HArg-X 11 -WSTPX 16 -WC iii (SEQ ID NO: 204);

[0082] Among them, X7, X8, X9, X 11 and X 16 As defined herein.

[0083] In yet another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0084] Ci -PF / 1-Nal-dD / dR-C ii -M / L-HArg / Lys-N / H / DWSTPI / P / HyP-WC iii (SEQ ID NO: 214).

[0085] In yet another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0086] C i -PF / 1-Nal-dD / dR-C ii -M / L-HArg-N / H / DWSTPI / P / HyP-WC iii (SEQ ID NO: 205).

[0087] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 8 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0088] C i -X6-X7-X8-C ii -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii (SEQ ID NO: 206)

[0089] Among them X6 to X8 and X 10 To X 17 As defined herein.

[0090] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 3 amino acids and the second of which consists of 8 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0091] C i -P-1-Nal-D / Cya-C ii -M-HArg / Cya-D / Cya-WSTP-HyP-WC ii(SEQ ID NO: 207). In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of the two loop sequences consisting of 3 amino acids and the second of the two loop sequences consisting of 9 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0092] C i -P / A / Hyp-F / YG / AC ii -X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii (SEQ ID NO:38);

[0093] wherein X1-X4 represent any amino acid residues, including modified and unnatural amino acids, Hyp represents hydroxyproline, 1-Nal represents 1-naphthylalanine, 2-Nal represents 2-naphthylalanine, and C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0094] In one embodiment, X1 is selected from the group consisting of M, Npg, HLeu, I, Q, and Nle; wherein Npg is neopentyl glycine, HLeu is homoleucine, and Nle is norleucine.

[0095] In one embodiment, X2 is selected from K, R, S, D, HArg, K(Ac) and A; wherein HArg is homoarginine, K(Ac) is N-acetylated lysine, and A represents the L- or D-isoform of alanine.

[0096] In one embodiment, X3 is selected from the group consisting of N, D, H and A; wherein A represents the L- or D-isoform of alanine.

[0097] In one embodiment, X4 is selected from W, D, T and A.

[0098] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 7 amino acids and the second of which consists of 3 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0099] C i -W / A / YP / ALD / S / AS / D / P / AYW / 1-Nal-C ii -X5-R / HArg / AIC iii(SEQ ID NO: 42);

[0100] Where X5 represents any amino acid residue, 1-Nal represents 1-naphthylalanine, HArg represents homoarginine, and C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0101] In one embodiment, X5 is selected from A, dA, G, dD, N, E and P.

[0102] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of 7 amino acids and the second of which consists of 3 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0103] C i -W / APLD / SS / DYWC ii -X5-RIC iii (SEQ ID NO: 39);

[0104] Where X5 represents any amino acid residue, and C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0105] In one embodiment, X5 is selected from A, G, D and N.

[0106] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, both of which consist of 6 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0107] C i -VTTSYDC- ii -F / WL / VH / R / TLL / GG / Q / HC iii (SEQ ID NO: 40);

[0108] Among them C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0109] In another embodiment, C i -P / A / Hyp-F / YG / AC ii-X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii The peptide ligand of (SEQ ID NO: 38) comprises an amino acid sequence selected from any one of SEQ ID NOS: 1-30:

[0110] C i PFGC ii MKNWSWPIWC iii (SEQ ID NO: 1);

[0111] C i PFGC ii MRNWSWPIWC iii (SEQ ID NO: 2);

[0112] C i PFGC ii (Npg)KNWSWPIWC iii (SEQ ID NO: 3);

[0113] C i PFGC ii (HLeu)KNWSWPIWC iii (SEQ ID NO: 4);

[0114] C i PFGC ii IKNWSWPIWC iii (SEQ ID NO: 5);

[0115] C i PFAC ii MKNWSWPIWC iii (SEQ ID NO: 6);

[0116] C i PFGC ii QKNWSWPIWC iii (SEQ ID NO:7);

[0117] C i PFGC ii MKNWSDPIWC iii (SEQ ID NO: 8);

[0118] C i PFGC ii MKNWSWPI(1-Nal)C iii (SEQ ID NO:9);

[0119] C i (Hyp)FGC ii MKNWSWPIWC iii (SEQ ID NO:10);

[0120] C i PYGC ii MKNWSWPIWC iii (SEQ ID NO:11);

[0121] C i PFGC ii MSNWSWPIWC iii (SEQ ID NO:12);

[0122] C i PFGC ii MDNWSWPIWC iii (SEQ ID NO:13);

[0123] C i PFGC ii M(HArg)NWSWPIWC iii (SEQ ID NO:14);

[0124] C i PFGC ii MKDWSWPIWC iii (SEQ ID NO:15);

[0125] C i PFGC ii MKHWSWPIWC iii (SEQ ID NO:16);

[0126] C i PFGC ii MKN(1-Nal)SWPIWC iii (SEQ ID NO:17);

[0127] C i PFGC ii MKN(2-Nal)SWPIWC iii (SEQ ID NO:18);

[0128] C i PFGC ii MKNWSTPIWC iii (SEQ ID NO:19);

[0129] C i PFGCii MKNWSWPDWC iii (SEQ ID NO:20);

[0130] C i PFGC ii MKNWSWPI(2-Nal)C iii (SEQ ID NO:21);

[0131] C i PFGC ii M(HArg)NWSWPIWC iii (SEQ ID NO:22);

[0132] C i PFGC ii M(K(Ac))NWSWPIWC iii (SEQ ID NO:23);

[0133] C i PFGC ii MKNWSAPIWC iii (SEQ ID NO:24);

[0134] C i PFGC ii MKNWSWPAWC iii (SEQ ID NO:25);

[0135] C i AFGC ii MKNWSWPIWC iii (SEQ ID NO:26);

[0136] C i PFGC ii MANWSWPIWC iii (SEQ ID NO:27);

[0137] C i PFGC ii MKAWSWPIWC iii (SEQ ID NO:28);

[0138] C i PFGC ii (Nle)KNWSWPIWC iii (SEQ ID NO:29); and

[0139] C i PFGC ii MKNWAWPIWCiii (SEQ ID NO:30);

[0140] wherein Ci, Cii and Ciii represent the first, second and third cysteine ​​residues, respectively, or pharmaceutically acceptable salts thereof.

[0141] In another embodiment, C i -P / A / Hyp-F / YG / AC ii -X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii The peptide ligand of (SEQ ID NO: 38) comprises an amino acid sequence selected from the group consisting of:

[0142] A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N002 or BCY428);

[0143] F1-A-(SQ ID NO: 1)-A (referred to herein as 80-09-02-N006);

[0144] Ac-(SEQ ID NO: 1) (referred to herein as 80-09-02-N008 or BCY7390);

[0145] Ac-[dD]-(SEQ ID NO: 1) (referred to herein as BCY7606);

[0146] A-(SEQ ID NO: 2)-A (referred to herein as 80-09-02-N003 or BCY429);

[0147] (1-Nal)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N009 or BCY7420);

[0148] (2-Nal)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N010 or BCY7421);

[0149] (33DPA)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N011 or BCY7422);

[0150] (44BPA)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N012 or BCY7521);

[0151] Ac-(SEQ ID NO: 3) (referred to herein as 80-09-02-N017);

[0152] Ac-(SEQ ID NO: 4) (referred to herein as 80-09-02-N018);

[0153] Ac-(SQ ID: 5) (referred to herein as 80-09-02-N019 or BCY7537);

[0154] Ac-(SEQ ID NO: 6) (referred to herein as 80-09-02-N020);

[0155] Ac-(SEQ ID NO: 7) (referred to herein as 80-09-02-N021 or BCY7539);

[0156] Ac-(SEQ ID NO:8) (referred to herein as 80-09-02-N022 or BCY7540);

[0157] Ac-(SEQ ID NO:9) (referred to herein as 80-09-02-N023);

[0158] Ac-(pCoF)-(SEQ ID NO: 1) (referred to herein as 80-09-02-N044);

[0159] Ac-(SEQ ID NO: 10) (referred to herein as 80-09-02-N045);

[0160] Ac-(SEQ ID NO: 11) (referred to herein as 80-09-02-N046 or BCY7657);

[0161] Ac-(SEQ ID NO: 12) (referred to herein as 80-09-02-N047 or BCY7658);

[0162] Ac-(SEQ ID NO: 13) (referred to herein as 80-09-02-N048 or BCY7659);

[0163] Ac-(SEQ ID NO: 14) (referred to herein as 80-09-02-N049);

[0164] Ac-(SEQ ID NO: 15) (referred to herein as 80-09-02-N050 or BCY7661);

[0165] SDN-(SEQ ID NO: 15)-A (referred to herein as BCY3387);

[0166] Ac-(SEQ ID NO: 16) (referred to herein as 80-09-02-N051 or BCY7662);

[0167] Ac-(SEQ ID NO: 17) (referred to herein as 80-09-02-N052);

[0168] Ac-(SEQ ID NO: 18) (referred to herein as 80-09-02-N053);

[0169] Ac-(SEQ ID NO: 19) (referred to herein as 80-09-02-N054 or BCY7665);

[0170] Ac-(SEQ ID NO: 20) (referred to herein as 80-09-02-N055 or BCY7666);

[0171] Ac-(SEQ ID NO: 21) (referred to herein as 80-09-02-N056);

[0172] A-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N001 or BCY3385);

[0173] A-(SEQ ID NO: 1)-TN(HArg) (referred to herein as 80-09-02-T01-N003 or BCY7281);

[0174] A-(SEQ ID NO: 1)-TN(DK) (referred to herein as 80-09-02-T01-N004 or BCY7282);

[0175] A-(SEQ ID NO: 22)-TNK (referred to herein as 80-09-02-T01-N005);

[0176] A-(SEQ ID NO: 23)-TNK (referred to herein as 80-09-02-T01-N006);

[0177] Ac-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N011 or BCY7391);

[0178] A-(SEQ ID NO: 24)-TNK (referred to herein as 80-09-02-T01-N012 or BCY7342);

[0179] A-(SEQ ID NO: 25)-TNK (referred to herein as 80-09-02-T01-N014 or BCY7344);

[0180] A-(SEQ ID NO: 1)-ANK (referred to herein as 80-09-02-T01-N016 or BCY7346);

[0181] A-(SEQ ID NO: 1)-[dA]NK (referred to herein as BCY7367);

[0182] A-(SEQ ID NO: 1)-TAK (referred to herein as 80-09-02-T01-N017 or BCY7347);

[0183] A-(SEQ ID NO: 1)-T[dA]K (referred to herein as BCY7368);

[0184] A-(SEQ ID NO: 1)-TNA (referred to herein as 80-09-02-T01-N018 or BCY7348);

[0185] A-(SEQ ID NO: 1)-TN[dA] (referred to herein as BCY7369);

[0186] Ac-[pCoPhe]-(SEQ ID NO: 1) (referred to herein as BCY7656);

[0187] A-(SEQ ID NO: 6)-TNK (referred to herein as 80-09-02-T01-N020 or BCY7354);

[0188] A-(SEQ ID NO: 26)-TNK (referred to herein as 80-09-02-T01-N022 or BCY7352);

[0189] A-(SEQ ID NO: 27)-TNK (referred to herein as 80-09-02-T01-N026 or BCY7356);

[0190] A-(SEQ ID NO: 28)-TNK (referred to herein as 80-09-02-T01-N027 or BCY7357);

[0191] A-(SEQ ID NO: 29)-TNK (referred to herein as 80-09-02-T01-N041 or BCY7372); and

[0192] A-(SEQ ID NO: 30)-TNK (referred to herein as 80-09-02-T01-N042 or BCY7424).

[0193] In another embodiment, C i -P / A / Hyp-F / YG / ACii -X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii The peptide ligand of (SEQ ID NO: 38) comprises an amino acid sequence selected from the group consisting of:

[0194] A-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N001 or BCY3385);

[0195] Ac-(SEQ ID NO: 1) (referred to herein as 80-09-02-N008 or BCY7390); and

[0196] Ac-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N011 or BCY7391).

[0197] The data are presented in Table 3 and demonstrate that the peptide ligands of this embodiment exhibit good levels of binding to human Bindex-4 (<100 nM) as demonstrated by SPR binding data.

[0198] In another embodiment, C i -W / APLD / SS / DYWC ii -X5-RIC iii The peptide ligand of (SEQ ID NO: 39) comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34:

[0199] C i WPLDSYWC ii ARIC iii (SEQ ID NO:31);

[0200] C i APLDDYWC ii GRIC iii (SEQ ID NO:32);

[0201] C i APLDDYWC ii DRIC iii (SEQ ID NO: 33); and

[0202] C i APLSDYWC ii NRIC iii (SEQ ID NO: 34);

[0203] Among them C i 、Cii and Cii i represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0204] In another embodiment, C i -W / APLD / SS / DYWC ii -C5-RIC iii The peptide ligand of (SEQ ID NO: 39) comprises an amino acid sequence selected from the group consisting of:

[0205] A-(SEQ ID NO: 31)-A (referred to herein as 80-10-00 or BCY488);

[0206] A-(SEQ ID NO: 32)-A (referred to herein as BCY432);

[0207] DDW-(SEQ ID NO: 32)-A (referred to herein as 80-10-11-T01 or BCY433);

[0208] VDW-(SEQ ID NO: 33)-A (referred to herein as 80-10-12-T01 or BCY462);

[0209] QKW-(SEQ ID NO: 34)-A (referred to herein as 80-10-13-T01 or BCY3400);

[0210] Q[HArg]W-(SEQ ID NO:34)-A (referred to herein as BCY7278);

[0211] Q[K(Ac)]W-(SEQ ID NO: 34)-A (referred to herein as BCY7280);

[0212] [Ac]QKW- (SEQ ID NO: 34) (referred to herein as BCY7392);

[0213] Q[dK]W-(SEQ ID NO: 34)-A (referred to herein as BCY7426);

[0214] Ac-AKW-(SEQ ID NO: 34) (referred to herein as BCY7622);

[0215] Ac-QAW- (SEQ ID NO: 34) (referred to herein as BCY7623);

[0216] Ac-QKA-(SEQ ID NO: 34) (referred to herein as BCY7624);

[0217] Ac-[dA]KW- (SEQ ID NO: 34) (referred to herein as BCY7634);

[0218] Ac-Q[dA]W- (SEQ ID NO: 34) (referred to herein as BCY7635);

[0219] Ac-QK[dA]-(SEQ ID NO: 34) (referred to herein as BCY7636);

[0220] Ac-Q[dD]W- (SEQ ID NO: 34) (referred to herein as BCY7993);

[0221] Ac-QK[1Nal]-(SEQ ID NO: 34) (referred to herein as BCY7996);

[0222] Ac-QK[2Nal]-(SEQ ID NO: 34) (referred to herein as BCY7997); and

[0223] Ac-(SEQ ID NO: 34) (referred to herein as BCY8044).

[0224] In another embodiment, C i -W / APLD / SS / DYWC ii -X5-RIC iii The peptide ligand of (SEQ ID NO: 39) comprises an amino acid sequence selected from the group consisting of:

[0225] [Ac]QKW- (SEQ ID NO: 34) (referred to herein as BCY7392).

[0226] The data are presented herein in Table 3, which demonstrates that the peptide ligands of this embodiment exhibit good levels of binding to human Bindex-4 (<100 nM), as demonstrated by SPR binding data.

[0227] In another embodiment, C i -W / A / YP / ALD / S / AS / D / P / AYW / 1-Nal-C ii -X5-R / HArg / AIC iii The peptide ligand of (SEQ ID NO: 42) comprises an amino acid sequence selected from the group consisting of:

[0228] CWPLDPYWCGRIC(SEQ ID NO:43);

[0229] CYPLSPYWCERIC(SEQ ID NO:44);

[0230] CWPLDDYWCPRIC(SEQ ID NO:58);

[0231] CAPLSDYWCN[HArg]IC(SEQ ID NO:65);

[0232] CAALSDYWCNRIC(SEQ ID NO:103);

[0233] CAPLADYWCNRIC(SEQ ID NO:104);

[0234] CAPLSAYWCNRIC(SEQ ID NO:105);

[0235] CAPLSDYWCARIC(SEQ ID NO:106);

[0236] CAPLSDYWCNAIC(SEQ ID NO:107);

[0237] CAPL[dA]DYWCNRIC(SEQ ID NO:108);

[0238] CAPLS[dA]YWCNRIC(SEQ ID NO:109);

[0239] CAPLSDYWC[dA]RIC(SEQ ID NO:110);

[0240] CAPLSDY[1Nal]CNRIC (SEQ ID NO: 153); and

[0241] CAPLSDYWC[dD]RIC(SEQ ID NO:154).

[0242] In another embodiment,

[0243] C i -W / A / YP / ALD / S / AS / D / P / AYW / 1-Nal-C ii -X5-R / HArg / AIC iii

[0244] The peptide ligand of (SEQ ID NO: 42) comprises an amino acid sequence selected from the group consisting of:

[0245] A-(SEQ ID NO:43)-A (hereinafter referred to as BCY430);

[0246] A-(SEQ ID NO:44)-A (hereinafter referred to as BCY431);

[0247] A-(SEQ ID NO: 58)-PQA (hereinafter referred to as BCY3401);

[0248] QKW-(SEQ ID NO:65)-A (hereinafter referred to as BCY7279);

[0249] Ac-QKW-(SEQ ID NO: 103) (hereinafter referred to as BCY7625);

[0250] Ac-QKW-(SEQ ID NO: 104) (hereinafter referred to as BCY7627);

[0251] Ac-QKW-(SEQ ID NO: 105) (hereinafter referred to as BCY7628);

[0252] Ac-QKW-(SEQ ID NO: 106) (hereinafter referred to as BCY7631);

[0253] Ac-QKW-(SEQ ID NO: 107) (hereinafter referred to as BCY7632);

[0254] Ac-QKW-(SEQ ID NO: 108) (hereinafter referred to as BCY7639);

[0255] Ac-QKW-(SEQ ID NO: 109) (hereinafter referred to as BCY7640);

[0256] Ac-QKW-(SEQ ID NO: 110) (hereinafter referred to as BCY7643);

[0257] Ac-QKW-(SEQ ID NO: 153) (hereinafter referred to as BCY7998); and

[0258] Ac-QKW-(SEQ ID NO: 154) (hereinafter referred to as BCY8000).

[0259] In another embodiment, C i -VTTSYDC ii -F / WL / VH / R / TLL / GG / Q / HC iii The peptide ligand of (SEQ ID NO: 40) comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-37:

[0260] C i VTTSYDC ii FLHLLGC iii(SEQ ID NO: 35);

[0261] C i VTTSYDC ii WVRLGQC iii (SEQ ID NO: 36); and

[0262] C i VTTSYDC ii WVTLGHC iii (SEQ ID NO: 37);

[0263] Among them C i 、C ii and C iii represent the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0264] In another embodiment, C i -VTTSYDC ii -F / WL / VH / R / TLL / GG / Q / HC iii The peptide ligand of (SEQ ID NO: 40) comprises an amino acid sequence selected from the group consisting of:

[0265] A-(SEQ ID NO:35)-A (referred to herein as 80-11-00 or BCY471);

[0266] A-(SEQ ID NO:36)-A (referred to herein as 80-11-01 or BCY472); and

[0267] A-(SEQ ID NO:37)-SRF (referred to herein as 80-11-08-T01 or BCY3406).

[0268] In yet another embodiment, C i -VTTSYDC ii -F / WL / VH / R / TLL / GG / Q / HC iii The peptide ligand of (SEQ ID NO: 40) comprises an amino acid sequence selected from the group consisting of:

[0269] Ac-(SEQ ID NO: 37)-SRF (referred to herein as BCY7393).

[0270] In another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X13 -X 14 -X 15 -X 16 -X 17 -C iii (SEQ ID NO: 41) The peptide ligand comprises an amino acid sequence selected from the following:

[0271] CPFGCMETWSWPIWC (SEQ ID NO: 45);

[0272] CPFGCMRGWSWPIWC (SEQ ID NO: 46);

[0273] CPFGCMSGWSWPIWC (SEQ ID NO: 47);

[0274] CPFGCMEGWSWPIWC (SEQ ID NO: 48);

[0275] CPFGCMEDWSWPIWC (SEQ ID NO: 49);

[0276] CPFGCMPGWSWPIWC (SEQ ID NO: 50);

[0277] CPFGCMKSWSWPIWC (SEQ ID NO: 51);

[0278] CPFGCMKTWSWPIWC (SEQ ID NO: 52);

[0279] CPFGCMKGWSWPIWC (SEQ ID NO: 53);

[0280] CPFGCQEHWSWPIWC (SEQ ID NO: 54);

[0281] CPFGClKSWSWPIWC (SEQ ID NO: 55);

[0282] CPFGCQEDWSWPIWC (SEQ ID NO: 56);

[0283] CPFGCMSDWSWPIWC (SEQ ID NO: 57);

[0284] CPFGCM[HArg]NWSWPIWC (SEQ ID NO: 59);

[0285] CPFGCM[K(Ac)]NWSWPIWC (SEQ ID NO: 60);

[0286] CPFGCM[K(Ac)]SWSWPIWC(SEQ ID NO:61);

[0287] CPFGC[Nle]KSWSWPIWC(SEQ ID NO:62);

[0288] CPFGCM[HArg]SWSWPIWC(SEQ ID NO:63);

[0289] CPFGCM[dK]SWSWPIWC(SEQ ID NO:64);

[0290] CP[dA]GCMKNWSWPIWC(SEQ ID NO:66);

[0291] CPF[dA]CMKNWSWPIWC(SEQ ID NO:67);

[0292] CPFGCM[dA]NWSWPIWC(SEQ ID NO:68);

[0293] CPFGCMK[dA]WSWPIWC(SEQ ID NO:69);

[0294] CPFGCMKN[dA]SWPIWC(SEQ ID NO:70);

[0295] CPFGCMKNWSWP[dA]WC(SEQ ID NO:71);

[0296] C[dA]FGCMKNWSWPIWC(SEQ ID NO:72);

[0297] CPFGC[tBuAla]KNWSWPIWC(SEQ ID NO:73);

[0298] CPFGC[HLeu]KNWSWPIWC(SEQ ID NO:74);

[0299] CPFGCMKNWSWPI[1Nal]C(SEQ ID NO:75);

[0300] CPF[dD]CM[HArg]NWSWPIWC(SEQ ID NO:76);

[0301] CPF[dA]CM[HArg]NWSWPIWC(SEQ ID NO:77);

[0302] CP[3MePhe]GCMKNWSWPIWC(SEQ ID NO:78);

[0303] CP[4MePhe]GCMKNWSWPIWC(SEQ ID NO:79);

[0304] CP[HPhe]GCMKNWSWPIWC(SEQ ID NO:80);

[0305] CPF[dD]CMKNWSWPIWC(SEQ ID NO:81);

[0306] CPFGC[Hse(Me)]KNWSWPIWC(SEQ ID NO:82);

[0307] CPFGCMKN[AzaTrp]SWPIWC(SEQ ID NO:83);

[0308] CPFGCMKNWSFPIWC(SEQ ID NO:84);

[0309] CPFGCMKNWSYPIWC(SEQ ID NO:85);

[0310] CPFGCMKNWS[1Nal]PIWC(SEQ ID NO:86);

[0311] CPFGCMKNWS[2Nal]PIWC(SEQ ID NO:87);

[0312] CPFGCMKNWS[AzaTrp]PIWC(SEQ ID NO:88);

[0313] CPFGCMKNWSW[Aze]IWC(SEQ ID NO:89);

[0314] CPFGCMKNWSW[Pip]IWC(SEQ ID NO:90);

[0315] CPFGCMKNWSWPIFC(SEQ ID NO:91);

[0316] CPFGCMKNWSWPIYC(SEQ ID NO:92);

[0317] CPFGCMKNWSWPI[AzaTrp]C(SEQ ID NO:93);

[0318] CGGFCMKNWSWPIWC(SEQ ID NO:94);

[0319] C[Aze]FGCMKNWSWPIWC(SEQ ID NO:95);

[0320] CPF[K(Ac)]CMKNWSWPIWC(SEQ ID NO:96);

[0321] CPFGCLKNWSWPIWC(SEQ ID NO:97);

[0322] CPFGC[MetO2]KNWSWPIWC(SEQ ID NO:98);

[0323] CPFGCMPNWSWPIWC(SEQ ID NO:99);

[0324] CPFGCMQNWSWPIWC(SEQ ID NO:100);

[0325] CPFGCMKNWSWPPWC(SEQ ID NO:101);

[0326] CP[2Pal]GCMKNWSWPIWC(SEQ ID NO:102);

[0327] CPFGCMKN[1Nal]SWPIWC(SEQ ID NO:111);

[0328] CPFGCMKN[2Nal]SWPIWC(SEQ ID NO:112);

[0329] CPFGCMKNWSWPI[2Nal]C(SEQ ID NO:113);

[0330] C[HyP]FGCMKNWSWPIWC(SEQ ID NO:114);

[0331] CPF[dD]CM[HArg]NWSTPIWC(SEQ ID NO:115);

[0332] CPF[dD]CM[HArg][dK]WSTPIWC(SEQ ID NO:116);

[0333] CPF[dD]CM[HArg]NWSTPKWC(SEQ ID NO:117);

[0334] C[Pro(4NH)]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:118);

[0335] CPF[dD]CMKNWSTPIWC(SEQ ID NO:119);

[0336] CPF[dK]CM[HArg]NWSTPIWC(SEQ ID NO:120);

[0337] CPF[dD]CK[HArg]NWSTPIWC(SEQ ID NO:121);

[0338] CPF[dD]CM[HArg]KWSTPIWC(SEQ ID NO:122);

[0339] C[Oxa]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:123);

[0340] CPF[dD]CM[HArg][Thi]WSTPIWC(SEQ ID NO:124);

[0341] CPF[dD]CM[HArg][4ThiAz]WSTPIWC(SEQ ID NO:125);

[0342] CPF[dD]CM[HArg][124TriAz]WSTPIWC(SEQ ID NO:126);

[0343] CPF[dD]CM[HArg]NWS[124TriAz]PIWC(SEQ ID NO:127);

[0344] CPF[dD]CM[HArg]NWST[Oxa]IWC(SEQ ID NO:128);

[0345] CP[DOPA][dD]CM[HArg]NWSTPIWC(SEQ ID NO:129);

[0346] CPF[dD]CM[HArg]NWS[DOPA]PlWC(SEQ ID NO:130);

[0347] CPF[dD]CM[HArg]NWS[THP(SO2)]PIWC(SEQ ID NO:131);

[0348] CPF[dD]CM[HArg]NWSTP[THP(SO2)]WC(SEQ ID NO:132);

[0349] CPF[dD]CM[HArg]N[5FTrp]STPIWC(SEQ ID NO:133);

[0350] CPF[dD]CM[HArg]NWSTPI[5FTrp]C(SEQ ID NO:134);

[0351] CPF[dD]CM[HArg]NWS[THP(O)]PIWC(SEQ ID NO:135);

[0352] CPF[dD]CM[HArg]NWSTP[THP(O)]WC(SEQ ID NO:136);

[0353] C[44DFP]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:137);

[0354] C[Oic]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:138);

[0355] CPF[dF]CM[HArg]NWSTPIWC(SEQ ID NO:139);

[0356] CPF[dE]CM[HArg]NWSTPIWC(SEQ ID NO:140);

[0357] CPF[dQ]CM[HArg]NWSTPIWC(SEQ ID NO:141);

[0358] CPF[dL]CM[HArg]NWSTPIWC(SEQ ID NO:142);

[0359] CPF[dS]CM[HArg]NWSTPIWC(SEQ ID NO:143);

[0360] CPF[dD]CM[HArg]NW[HSer]TPIWC(SEQ ID NO:144);

[0361] CPF[dD]CM[HArg]NWSTP[C5A]WC(SEQ ID NO:145);

[0362] CPF[dD]CM[HArg]NWSTP[Cpa]WC(SEQ ID NO:146);

[0363] CPF[dD]CM[HArg]NWSTP[Cha]WC(SEQ ID NO:147);

[0364] CPF[dD]C[HGln][HArg]NWSTPIWC(SEQ ID NO:148);

[0365] CPF[dD]C[C5A][HArg]NWSTPIWC(SEQ ID NO:149);

[0366] CPF[dD]CM[HArg]N[Trp(Me)]STPIWC(SEQ ID NO:150);

[0367] CPF[dD][NMeCys]M[HArg]NWSTPIWC(SEQ ID NO:151);

[0368] CPF[dD]C[HArg]NWS[NMeThr]PIWC(SEQ ID NO:152);

[0369] CP[1Nal][dD]CM[HArg]NWSTPIWC(SEQ ID NO:155);

[0370] CP[2Nal][dD]CM[HArg]NWSTPIWC(SEQ ID NO:156);

[0371] CP[44BPA][dD]CM[HArg]NWSTPIWC(SEQ ID NO:157);

[0372] CPF[dD]CM[HArg]NWSTPPWC(SEQ ID NO:158);

[0373] CPF[dD]CM[HArg]NWSTP[HyP]WC(SEQ ID NO:159);

[0374] CPF[dD]CL[HArg]NWSTPPWC(SEQ ID NO:160);

[0375] CPF[dD]CL[HArg]NWSTPIWC(SEQ ID NO:161);

[0376] CPY[dD]CM[HArg]NWSTPIWC(SEQ ID NO:162);

[0377] C[Aib]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:163);

[0378] C[Sar]F[dD]CM[HArg]NWSTPIWC(SEQ ID NO:164);

[0379] CPF[dR]CM[HArg]NWSTPIWC(SEQ ID NO:165);

[0380] CPF[dD]CM[HArg]NWSTPKWC(SEQ ID NO:166);

[0381] CP[1Nal][dD]CM[HArg]NWSTP[HyP]WC(SEQ ID NO:167);

[0382] CP[1Nal][dD]CM[HArg]HWSTP[HyP]WC(SEQ ID NO:168):

[0383] CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC(SEQ ID NO:169);

[0384] CP[1Nal][dD]CM[HArg]DWSTPIWC(SEQ ID NO:170);

[0385] CP[1Nal][dR]CM[HArg]NWSTP[HyP]WC(SEQ ID NO:171);

[0386] CP[1Nal][dR]CM[HArg]HWSTP[HyP]WC(SEQ ID NO:172);

[0387] CPF[dD]CM[NMeHArg]NWSTPIWC(SEQ ID NO:173);

[0388] CPF[dD]CM[HArg][NMeAsn]WSTPIWC(SEQ ID NO:174);

[0389] CPF[dD]CM[HArg]NWS[NMeThr]PIWC(SEQ ID NO:175);

[0390] CPF[dD]CM[HArg]NWSTP[NMelle]WC(SEQ ID NO:176);

[0391] CP[1Nal][dD]CM[HArg][Cya]WSTP[HyP]WC(SEQ ID NO:177);

[0392] CP[1Nal][dD]CM[Cya]DWSTP[HyP]WC(SEQ ID NO:178);

[0393] CP[1Nal][DCya]CM[HArg]DWSTP[HyP]WC(SEQ ID NO:179);

[0394] CP[1Nal][dD]CM[HArg]DWDTP[HyP]WC(SEQ ID NO:180);

[0395] CP[2Nal][dD]CM[HArg]DWSTP[HyP]WC(SEQ ID NO:181);

[0396] CP[1Nal][dD]CM[HArg]DWTTP[HyP]WC(SEQ ID NO:182);

[0397] CP[1Nal][dD]CM[HArg]DW[HSer]TP[HyP]WC(SEQ ID NO:183);

[0398] CP[1Nal][dD]CM[HArg]DW[dS]TP[HyP]WC(SEQ ID NO:184);

[0399] CP[1Nal][dD]CM[HArg]DWSSP[HyP]WC(SEQ ID NO:185);

[0400] CP[1Nal][dD]CM[Agb]DWSTP[HyP]WC(SEQ ID NO:186);

[0401] CP[1Nal][dD]CMPDWSTP[HyP]WC(SEQ ID NO:187);

[0402] CP[1Nal][dD]CM[HyP]DWSTP[HyP]WC(SEQ ID NO:188);

[0403] CP[1Nal][dR]CM[HArg]DWSTP[HyP]WC(SEQ ID NO:189);

[0404] CP[1Nal][dR]CM[HArg]DWDTP[HyP]WC(SEQ ID NO:190);

[0405] CP[2Nal][dR]CM[HArg]DWSTP[HyP]WC(SEQ ID NO:191);

[0406] CP[1Nal][dR]CM[HArg]DWTTP[HyP]WC(SEQ ID NO:192);

[0407] CP[1Nal][dR]CM[HArg]DW[HSer]TP[HyP]WC(SEQ ID NO:193);

[0408] CP[1Nal][dR]CM[HArg]DW[dS]TP[HyP]WC(SEQ ID NO:194);

[0409] CP[1Nal][dR]CM[HArg]DWSSP[HyP]WC(SEQ ID NO:195);

[0410] CP[1Nal][dR]CM[Agb]DWSTP[HyP]WC(SEQ ID NO:196);

[0411] CP[1Nal][dR]CMPDWSTP[HyP]WC(SEQ ID NO:197);

[0412] CP[1Nal][dR]CM[HyP]DWSTP[HyP]WC(SEQ ID NO:198);

[0413] CP[1Nal][dD]CL[HArg]DWSTPIWC(SEQ ID NO:199);

[0414] CP[1Nal][dD]CL[HArg]DWSTP[HyP]WC(SEQ ID NO:200);

[0415] CP[1Nal][dR]CL[HArg]DWSTP[HyP]WC(SEQ ID NO:201);

[0416] CP[1Nal][dR]CL[HArg]HWSTP[HyP]WC(SEQ ID NO:202);

[0417] CP[1Nal][dR]CM[HArg]DWSTPIWC(SEQ ID NO:203);

[0418] CP[1Nal][DCya]CM[Cya]DWSTP[HyP]WC (SEQ ID NO:208);

[0419] CP[1Nal][DCya]CM[HArg][Cya]WSTP[HyP]WC (SEQ ID NO:209);

[0420] CP[1Nal][dD]CM[Cya][Cya]WSTP[HyP]WC (SEQ ID NO:210);

[0421] CP[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:211);

[0422] CP[1Nal][dD1CMKDWSTP[HyP]WC(SEQ ID NO:212);

[0423] CP[1Nal][dD]CM[HArg]D[dW]STP[HyP][dW]C (SEQ ID NO: 215); and

[0424] CPFGCM[HArg]DWSTP[HyP]WC (SEQ ID NO:216).

[0425] In another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0426] CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 169).

[0427] In one embodiment, the peptide ligand of the present invention is a peptide ligand that is not the amino acid sequence CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 169).

[0428] In another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0429] DDA-(SEQ ID NO: 45)-A (hereinafter referred to as BCY3386);

[0430] DTA-(SEQ ID NO: 46)-A (hereinafter referred to as BCY3388);

[0431] DSE-(SEQ ID NO: 47)-A (hereinafter referred to as BCY3389);

[0432] HDA-(SEQ ID NO: 48)-A (hereinafter referred to as BCY3390);

[0433] MDT-(SEQ ID NO: 49)-A (hereinafter referred to as BCY3391);

[0434] DPG-(SEQ ID NO: 50)-A (hereinafter referred to as BCY3392);

[0435] HDS-(SEQ ID NO: 51)-A (hereinafter referred to as BCY3393);

[0436] (DH)DS-(SEQ ID NO: 51)-A (hereinafter referred to as BCY7272);

[0437] A-(SEQ ID NO: 52)-TDK (hereinafter referred to as BCY3394);

[0438] A-(SEQ ID NO: 53)-LKD (hereinafter referred to as BCY3395);

[0439] A-(SEQ ID NO: 54)-TTA (hereinafter referred to as BCY3396);

[0440] A-(SEQ ID NO: 55)-QME (hereinafter referred to as BCY3397);

[0441] A-(SEQ ID NO: 56)-LSE (hereinafter referred to as BCY3398); A-(SEQ ID NO: 57)-STD (hereinafter referred to as BCY3399);

[0442] A-(SEQ ID NO: 59)-TNK (hereinafter referred to as BCY7265);

[0443] Ac-(SEQ ID NO:59) (hereinafter referred to as BCY7660);

[0444] A-(SEQ ID NO:60)-TNK (hereinafter referred to as BCY7266);

[0445] Ac-(SEQ ID NO:60) (hereinafter referred to as BCY7616);

[0446] HDS-(SEQ ID NO:61)-A (hereinafter referred to as BCY7273);

[0447] HDS-(SEQ ID NO: 62)-A (hereinafter referred to as BCY7274);

[0448] HDS-(SEQ ID NO: 63)-A (hereinafter referred to as BCY7275);

[0449] HDS-(SEQ ID NO: 64)-A (hereinafter referred to as BCY7276);

[0450] A-(SEQ ID NO:66)-TNK (hereinafter referred to as BCY7349);

[0451] A-(SEQ ID NO: 67)-TNK (hereinafter referred to as BCY7350);

[0452] Ac-(SEQ ID NO:67) (hereinafter referred to as BCY7538);

[0453] A-(SEQ ID NO: 68)-TNK (hereinafter referred to as BCY7359);

[0454] A-(SEQ ID NO:69)-TNK (hereinafter referred to as BCY7360);

[0455] A-(SEQ ID NO:70)-TNK (hereinafter referred to as BCY7361);

[0456] A-(SEQ ID NO:71)-TNK (hereinafter referred to as BCY7365);

[0457] A-(SEQ ID NO:72)-TNK (hereinafter referred to as BCY7370);

[0458] Ac-(SEQ ID NO:73) (hereinafter referred to as BCY7535);

[0459] Ac-(SEQ ID NO:74) (hereinafter referred to as BCY7536);

[0460] Ac-(SEQ ID NO:75) (hereinafter referred to as BCY7541);

[0461] [B-Ala][Sar5]-(SEQ ID NO:76) (hereinafter referred to as BCY7556);

[0462] Ac-[B-Ala][Sar5]-(SEQ ID NO:76) (hereinafter referred to as BCY7558);

[0463] [B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7557);

[0464] Ac-[B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7559);

[0465] Ac-(SEQ ID NO:78) (hereinafter referred to as BCY7580);

[0466] Ac-(SEQ ID NO:79) (hereinafter referred to as BCY7581);

[0467] Ac-(SEQ ID NO:80) (hereinafter referred to as BCY7582);

[0468] Ac-(SEQ ID NO:81) (hereinafter referred to as BCY7584);

[0469] Ac-(SEQ ID NO:82) (hereinafter referred to as BCY7585);

[0470] Ac-(SEQ ID NO:83) (hereinafter referred to as BCY7588);

[0471] Ac-(SEQ ID NO:84) (hereinafter referred to as BCY7589);

[0472] Ac-(SEQ ID NO:85) (hereinafter referred to as BCY7590);

[0473] Ac-(SEQ ID NO:86) (hereinafter referred to as BCY7591);

[0474] Ac-(SEQ ID NO:87) (hereinafter referred to as BCY7592);

[0475] Ac-(SEQ ID NO:88) (hereinafter referred to as BCY7593);

[0476] Ac-(SEQ ID NO:89) (hereinafter referred to as BCY7594);

[0477] Ac-(SEQ ID NO:90) (hereinafter referred to as BCY7595);

[0478] Ac-(SEQ ID NO:91) (hereinafter referred to as BCY7596);

[0479] Ac-(SEQ ID NO:92) (hereinafter referred to as BCY7597);

[0480] Ac-(SEQ ID NO:93) (hereinafter referred to as BCY7598);

[0481] Ac-(SEQ ID NO:94) (hereinafter referred to as BCY7607);

[0482] Ac-(SEQ ID NO:95) (hereinafter referred to as BCY7608);

[0483] Ac-(SEQ ID NO:96) (hereinafter referred to as BCY7611);

[0484] Ac-(SEQ ID NO:97) (hereinafter referred to as BCY7612);

[0485] Ac-(SEQ ID NO:98) (hereinafter referred to as BCY7613);

[0486] Ac-(SEQ ID NO:99) (hereinafter referred to as BCY7614);

[0487] Ac-(SEQ ID NO: 100) (hereinafter referred to as BCY7615);

[0488] Ac-(SEQ ID NO: 101) (hereinafter referred to as BCY7618);

[0489] Ac-(SEQ ID NO: 102) (hereinafter referred to as BCY7620);

[0490] Ac-(SEQ ID NO: 111) (hereinafter referred to as BCY7663);

[0491] Ac-(SEQ ID NO: 112) (hereinafter referred to as BCY7664);

[0492] Ac-(SEQ ID NO: 113) (hereinafter referred to as BCY7667);

[0493] Ac-(SEQ ID NO: 114) (hereinafter referred to as BCY7668);

[0494] Ac-(SEQ ID NO: 115) (hereinafter referred to as BCY7765);

[0495] (SEQ ID NO: 115) (hereinafter referred to as BCY7793);

[0496] (MeO-dPEG12) (SEQ ID NO: 115) (hereinafter referred to as BCY8087);

[0497] (carboxyfluorescein) (SEQ ID NO: 115) (hereinafter referred to as BCY8208);

[0498] (PEG3)(PEG3)(SEQ ID NO:115)(hereinafter referred to as BCY7815);

[0499] (MeO-dPEG12)(PEG3)(PEG3) (SEQ ID NO: 115) (hereinafter referred to as BCY8094);

[0500] Ac-DDD-(SEQ ID NO: 115) (hereinafter referred to as BCY8028);

[0501] Ac-[dD][dD][dD]-(SEQ ID NO: 115) (hereinafter referred to as BCY8029);

[0502] [B-Ala][Sar10]-(SEQ ID NO: 115) (hereinafter referred to as BCY7814);

[0503] Ac-(SEQ ID NO: 116) (hereinafter referred to as BCY7816);

[0504] Ac-(SEQ ID NO: 116) (MeO-dPEG12) linked to D-Lys6 (hereinafter referred to as BCY8084);

[0505] Ac-(SEQ ID NO: 117) (hereinafter referred to as BCY7817);

[0506] Ac-(SEQ ID NO: 118) (hereinafter referred to as BCY7818;

[0507] Ac-(SEQ ID NO: 118)(MeO-dPEG12) is linked to Pro(4NH)1 (hereinafter referred to as BCY8086);

[0508] Ac-(SEQ ID NO: 119) (hereinafter referred to as BCY7819;

[0509] Ac-(SEQ ID NO: 119)(MeO-dPEG12) linked to Lys5 (hereinafter referred to as BCY8088);

[0510] Ac-(SEQ ID NO: 120) (hereinafter referred to as BCY7820;

[0511] Ac-(SEQ ID NO: 120)(MeO-dPEG12) linked to D-Lys3 (hereinafter referred to as BCY8089);

[0512] Ac-(SEQ ID NO: 121) (hereinafter referred to as BCY7821);

[0513] Ac-(SEQ ID NO: 121)(MeO-dPEG12) linked to Lys4 (hereinafter referred to as BCY8090);

[0514] Ac-(SEQ ID NO: 122) (hereinafter referred to as BCY7822);

[0515] Ac-(SEQ ID NO: 122)(MeO-dPEG12) linked to Lys6 (hereinafter referred to as BCY8091);

[0516] Ac-(SEQ ID NO: 123) (hereinafter referred to as BCY7876);

[0517] Ac-(SEQ ID NO: 124) (hereinafter referred to as BCY7877);

[0518] Ac-(SEQ ID NO: 125) (hereinafter referred to as BCY7879);

[0519] Ac-(SEQ ID NO: 126) (hereinafter referred to as BCY7881);

[0520] Ac-(SEQ ID NO: 127) (hereinafter referred to as BCY7883);

[0521] Ac-(SEQ ID NO: 128) (hereinafter referred to as BCY7884);

[0522] Ac-(SEQ ID NO: 129) (hereinafter referred to as BCY7886);

[0523] Ac-(SEQ ID NO: 130) (hereinafter referred to as BCY7887);

[0524] Ac-(SEQ ID NO: 131) (hereinafter referred to as BCY7889);

[0525] Ac-(SEQ ID NO: 132) (hereinafter referred to as BCY7890);

[0526] Ac-(SEQ ID NO: 133) (hereinafter referred to as BCY7891);

[0527] Ac-(SEQ ID NO: 134) (hereinafter referred to as BCY7892);

[0528] Ac-(SEQ ID NO: 135) (hereinafter referred to as BCY7894);

[0529] Ac-(SEQ ID NO: 136) (hereinafter referred to as BCY7895);

[0530] Ac-(SEQ ID NO: 137) (hereinafter referred to as BCY7896);

[0531] Ac-(SEQ ID NO: 138) (hereinafter referred to as BCY7897);

[0532] Ac-(SEQ ID NO: 139) (hereinafter referred to as BCY7902);

[0533] Ac-(SEQ ID NO: 140) (hereinafter referred to as BCY7903);

[0534] Ac-(SEQ ID NO: 141) (hereinafter referred to as BCY7904);

[0535] Ac-(SEQ ID NO: 142) (hereinafter referred to as BCY7906);

[0536] Ac-(SEQ ID NO: 143) (hereinafter referred to as BCY7907);

[0537] Ac-(SEQ ID NO: 144) (hereinafter referred to as BCY7908);

[0538] Ac-(SEQ ID NO: 145) (hereinafter referred to as BCY7911);

[0539] Ac-(SEQ ID NO: 146) (hereinafter referred to as BCY7912);

[0540] Ac-(SEQ ID NO: 147) (hereinafter referred to as BCY7913);

[0541] Ac-(SEQ ID NO: 148) (hereinafter referred to as BCY7914);

[0542] Ac-(SEQ ID NO: 149) (hereinafter referred to as BCY7915);

[0543] Ac-(SEQ ID NO: 150) (hereinafter referred to as BCY7916);

[0544] Ac-(SEQ ID NO: 151) (hereinafter referred to as BCY7973);

[0545] Ac-(SEQ ID NO: 152) (hereinafter referred to as BCY7979);

[0546] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[0547] Ac-(SEQ ID NO: 156) (hereinafter referred to as BCY8031);

[0548] Ac-(SEQ ID NO: 157) (hereinafter referred to as BCY8032);

[0549] Ac-(SEQ ID NO: 158) (hereinafter referred to as BCY8036);

[0550] Ac-(SEQ ID NO: 159) (hereinafter referred to as BCY8037);

[0551] Ac-(SEQ ID NO: 160) (hereinafter referred to as BCY8038);

[0552] Ac-(SEQ ID NO: 161) (hereinafter referred to as BCY8039);

[0553] Ac-(SEQ ID NO: 162) (hereinafter referred to as BCY8040);

[0554] Ac-(SEQ ID NO: 163) (hereinafter referred to as BCY8041);

[0555] Ac-(SEQ ID NO: 164) (hereinafter referred to as BCY8042);

[0556] Ac-(SEQ ID NO: 165) (hereinafter referred to as BCY8042);

[0557] Ac-(SEQ ID NO: 166) (hereinafter referred to as BCY8085);

[0558] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[0559] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[0560] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[0561] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[0562] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[0563] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[0564] (SEQ ID NO: 169) (hereinafter referred to as BCY8116);

[0565] Fluorescein-(SEQ ID NO: 169) (hereinafter referred to as BCY8205);

[0566] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234);

[0567] [PYA][B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8846);

[0568] Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[0569] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[0570] (SEQ ID NO: 170) (hereinafter referred to as BCY8206);

[0571] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[0572] (SEQ ID NO: 171) (hereinafter referred to as BCY8207);

[0573] [B-Ala][Sar10]-(SEQ ID NO: 171) (hereinafter referred to as BCY8232);

[0574] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[0575] Ac-(SEQ ID NO: 173) (hereinafter referred to as BCY8153);

[0576] Ac-(SEQ ID NO: 174) (hereinafter referred to as BCY8154);

[0577] Ac-(SEQ ID NO: 175) (hereinafter referred to as BCY8157);

[0578] Ac-(SEQ ID NO: 176) (hereinafter referred to as BCY8158);

[0579] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[0580] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[0581] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[0582] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[0583] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[0584] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[0585] [B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8269);

[0586] Ac-(SEQ ID NO: 180) (hereinafter referred to as BCY8174);

[0587] Ac-(SEQ ID NO: 181) (hereinafter referred to as BCY8175);

[0588] Ac-(SEQ ID NO: 182) (hereinafter referred to as BCY8176);

[0589] Ac-(SEQ ID NO: 183) (hereinafter referred to as BCY8177);

[0590] Ac-(SEQ ID NO: 184) (hereinafter referred to as BCY8178);

[0591] Ac-(SEQ ID NO: 185) (hereinafter referred to as BCY8180);

[0592] Ac-(SEQ ID NO: 186) (hereinafter referred to as BCY8181);

[0593] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[0594] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[0595] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[0596] [B-Ala][Sar10]-(SEQ ID NO: 189) (hereinafter referred to as BCY8235);

[0597] Ac-(SEQ ID NO: 190) (hereinafter referred to as BCY8185);

[0598] Ac-(SEQ ID NO: 191) (hereinafter referred to as BCY8186);

[0599] Ac-(SEQ ID NO: 192) (hereinafter referred to as BCY8187);

[0600] Ac-(SEQ ID NO: 193) (hereinafter referred to as BCY8188);

[0601] Ac-(SEQ ID NO: 194) (hereinafter referred to as BCY8189);

[0602] Ac-(SEQ ID NO: 195) (hereinafter referred to as BCY8191);

[0603] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[0604] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[0605] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[0606] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[0607] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[0608] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[0609] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[0610] [B-Ala][Sar10]-(SEQ ID NO: 202) (hereinafter referred to as BCY8231);

[0611] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[0612] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[0613] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280);

[0614] [B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8273);

[0615] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281);

[0616] Ac-(SEQ ID NO: 211) (hereinafter referred to as BCY8831);

[0617] [B-Ala][Sar10]-(SEQ ID NO: 212) (hereinafter referred to as BCY8238);

[0618] (SEQ ID NO: 215) (hereinafter referred to as BCY11415);

[0619] [PYA][B-Ala][Sar10]-(SEQ ID NO: 215) (hereinafter referred to as BCY11942); and

[0620] (SEQ ID NO: 216) (hereinafter referred to as BCY11414).

[0621] In another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0622] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[0623] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[0624] (SEQ ID NO: 169) (hereinafter referred to as BCY8116);

[0625] Fluorescein-(SEQ ID NO: 169) (hereinafter referred to as BCY8205); and

[0626] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234).

[0627] In one embodiment, the peptide ligand of the present invention is a peptide ligand other than the following amino acid sequence:

[0628] [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234);

[0629] Ac-[B-Ala][Sar5]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);

[0630] Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126);

[0631] (SEQ ID NO: 1) (hereinafter referred to as BCY8116);

[0632] Fluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205); and

[0633] [PYA][B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846).

[0634] In another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0635] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234).

[0636] In yet another embodiment, C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0637] Ac-[B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7559);

[0638] Ac-(SEQ ID NO: 101) (hereinafter referred to as BCY7618);

[0639] Ac-(SEQ ID NO: 115) (hereinafter referred to as BCY7765);

[0640] (SEQ ID NO: 115) (hereinafter referred to as BCY7793);

[0641] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[0642] Ac-(SEQ ID NO: 160) (hereinafter referred to as BCY8038);

[0643] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[0644] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[0645] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[0646] Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[0647] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[0648] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[0649] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[0650] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[0651] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[0652] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[0653] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[0654] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[0655] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[0656] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[0657] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[0658] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[0659] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[0660] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[0661] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[0662] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[0663] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[0664] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[0665] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[0666] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[0667] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[0668] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[0669] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[0670] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[0671] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280); and

[0672] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281).

[0673] The data are presented in Table 3 and demonstrate that the peptide ligands of this embodiment exhibit good levels of binding to human Bindex-4 (<100 nM) as demonstrated by SPR binding data.

[0674] In one embodiment, the C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii The peptide ligand of (SEQ ID NO: 41) comprises an amino acid sequence selected from the group consisting of:

[0675] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[0676] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[0677] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[0678] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[0679] Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[0680] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[0681] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[0682] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[0683] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[0684] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[0685] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[0686] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[0687] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[0688] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[0689] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[0690] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[0691] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[0692] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[0693] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[0694] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[0695] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[0696] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[0697] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[0698] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[0699] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[0700] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[0701] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[0702] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[0703] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[0704] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280); and

[0705] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281).

[0706] The data presented herein in Table 3 demonstrate that the peptide ligands of this embodiment exhibit excellent levels of binding to human Bindex-4 (<10 nM) as demonstrated by SPR binding data.

[0707] In one embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0708] A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N002 or BCY428);

[0709] Fl-A-(SQ ID NO:1)-A (referred to herein as 80-09-02-N006);

[0710] Ac-(SEQ ID NO: 1) (referred to herein as 80-09-02-N008 or BCY7390);

[0711] Ac-[dD]-(SEQ ID NO: 1) (referred to herein as BCY7606);

[0712] A-(SEQ ID NO:2)-A (referred to herein as 80-09-02-N003 or BCY429);

[0713] (1-Nal)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N009 or BCY7420);

[0714] (2-Nal)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N010 or BCY7421);

[0715] (33DPA)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N011 or BCY7422);

[0716] (44BPA)A-(SEQ ID NO: 1)-A (referred to herein as 80-09-02-N012 or BCY7521);

[0717] Ac-(SEQ ID NO:3) (referred to herein as 80-09-02-N017);

[0718] Ac-(SEQ ID NO:4) (referred to herein as 80-09-02-N018);

[0719] Ac-(SQ ID NO:5) (referred to herein as 80-09-02-N019 or BCY7537);

[0720] Ac-(SEQ ID NO:6) (referred to herein as 80-09-02-N020);

[0721] Ac-(SEQ ID NO:7) (referred to herein as 80-09-02-N021 or BCY7539);

[0722] Ac-(SEQ ID NO:8) (referred to herein as 80-09-02-N022 or BCY7540);

[0723] Ac-(SEQ ID NO:9) (referred to herein as 80-09-02-N023);

[0724] Ac-(pCoF)-(SEQ ID NO: 1) (referred to herein as 80-09-02-N044);

[0725] Ac-(SEQ ID NO: 10) (referred to herein as 80-09-02-N045);

[0726] Ac-(SEQ ID NO: 11) (referred to herein as 80-09-02-N046 or BCY7657);

[0727] Ac-(SEQ ID NO: 12) (referred to herein as 80-09-02-N047 or BCY7658);

[0728] Ac-(SEQ ID NO: 13) (referred to herein as 80-09-02-N048 or BCY7659);

[0729] Ac-(SEQ ID NO: 14) (referred to herein as 80-09-02-N049);

[0730] Ac-(SEQ ID NO: 15) (referred to herein as 80-09-02-N050 or BCY7661);

[0731] SDN-(SEQ ID NO: 15)-A (referred to herein as BCY3387);

[0732] Ac-(SEQ ID NO: 16) (referred to herein as 80-09-02-N051 or BCY7662);

[0733] Ac-(SEQ ID NO: 17) (referred to herein as 80-09-02-N052);

[0734] Ac-(SEQ ID NO: 18) (referred to herein as 80-09-02-N053);

[0735] Ac-(SEQ ID NO: 19) (referred to herein as 80-09-02-N054 or BCY7665);

[0736] Ac-(SEQ ID NO:20) (referred to herein as 80-09-02-N055 or BCY7666);

[0737] Ac-(SEQ ID NO:21) (referred to herein as 80-09-02-N056);

[0738] A-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N001 or BCY3385);

[0739] A-(SEQ ID NO: 1)-TN(HArg) (referred to herein as 80-09-02-T01-N003 or BCY7281);

[0740] A-(SEQ ID NO: 1)-TN(DK) (referred to herein as 80-09-02-T01-N004 or BCY7282);

[0741] A-(SEQ ID NO:22)-TNK (referred to herein as 80-09-02-T01-N005);

[0742] A-(SEQ ID NO:23)-TNK (referred to herein as 80-09-02-T01-N006);

[0743] Ac-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N011 or BCY7391);

[0744] A-(SEQ ID NO:24)-TNK (referred to herein as 80-09-02-T01-N012 or BCY7342);

[0745] A-(SEQ ID NO:25)-TNK (referred to herein as 80-09-02-T01-N014 or BCY7344);

[0746] A-(SEQ ID NO: 1)-ANK (referred to herein as 80-09-02-T01-N016 or BCY7346);

[0747] A-(SEQ ID NO: 1)-[dA]NK (referred to herein as BCY7367);

[0748] A-(SEQ ID NO: 1)-TAK (referred to herein as 80-09-02-T01-N017 or BCY7347);

[0749] A-(SEQ ID NO: 1)-T[dA]K (referred to herein as BCY7368);

[0750] A-(SEQ ID NO: 1)-TNA (referred to herein as 80-09-02-T01-N018 or BCY7348);

[0751] A-(SEQ ID NO: 1)-TN[dA] (referred to herein as BCY7369);

[0752] Ac-[pCoPhe]-(SEQ ID NO: 1) (referred to herein as BCY7656);

[0753] A-(SEQ ID NO:6)-TNK (referred to herein as 80-09-02-T01-N020 or BCY7354);

[0754] A-(SEQ ID NO:26)-TNK (referred to herein as 80-09-02-T01-N022 or BCY7352);

[0755] A-(SEQ ID NO:27)-TNK (referred to herein as 80-09-02-T01-N026 or BCY7356);

[0756] A-(SEQ ID NO:28)-TNK (referred to herein as 80-09-02-T01-N027 or BCY7357);

[0757] A-(SEQ ID NO:29)-TNK (referred to herein as 80-09-02-T01-N041 or BCY7372);

[0758] A-(SEQ ID NO:30)-TNK (referred to herein as 80-09-02-T01-N042 or BCY7424);

[0759] A-(SEQ ID NO:31)-A (referred to herein as 80-10-00 or BCY488);

[0760] A-(SEQ ID NO:32)-A (referred to herein as BCY432);

[0761] DDW-(SEQ ID NO:32)-A (referred to herein as 80-10-11-T01 or BCY433);

[0762] VDW-(SEQ ID NO:33)-A (referred to herein as 80-10-12-T01 or BCY462);

[0763] QKW-(SEQ ID NO:34)-A (referred to herein as 80-10-13-T01 or BCY3400);

[0764] Q[HArg]W-(SEQ ID NO:34)-A (referred to herein as BCY7278);

[0765] Q[K(Ac)]W-(SEQ ID NO:34)-A (referred to herein as BCY7280);

[0766] [Ac]QKW-(SEQ ID NO:34) (referred to herein as BCY7392);

[0767] Q[dK]W-(SEQ ID NO:34)-A (referred to herein as BCY7426);

[0768] Ac-AKW-(SEQ ID NO:34) (referred to herein as BCY7622);

[0769] Ac-QAW-(SEQ ID NO:34) (referred to herein as BCY7623);

[0770] Ac-QKA-(SEQ ID NO:34) (referred to herein as BCY7624);

[0771] Ac-[dA]KW-(SEQ ID NO:34) (referred to herein as BCY7634);

[0772] Ac-Q[dA]W- (SEQ ID NO: 34) (referred to herein as BCY7635);

[0773] Ac-QK[dA]-(SEQ ID NO:34) (referred to herein as BCY7636);

[0774] Ac-Q[dD]W- (SEQ ID NO: 34) (referred to herein as BCY7993);

[0775] Ac-QK[1Nal]-(SEQ ID NO:34) (referred to herein as BCY7996);

[0776] Ac-QK[2Nal]-(SEQ ID NO:34) (referred to herein as BCY7997);

[0777] Ac-(SEQ ID NO:34) (referred to herein as BCY8044);

[0778] A-(SEQ ID NO:43)-A (hereinafter referred to as BCY430);

[0779] A-(SEQ ID NO:44)-A (hereinafter referred to as BCY431);

[0780] A-(SEQ ID NO: 58)-PQA (hereinafter referred to as BCY3401);

[0781] QKW-(SEQ ID NO:65)-A (hereinafter referred to as BCY7279);

[0782] Ac-QKW-(SEQ ID NO: 103) (hereinafter referred to as BCY7625);

[0783] Ac-QKW-(SEQ ID NO: 104) (hereinafter referred to as BCY7627);

[0784] Ac-QKW-(SEQ ID NO: 105) (hereinafter referred to as BCY7628);

[0785] Ac-QKW-(SEQ ID NO: 106) (hereinafter referred to as BCY7631);

[0786] Ac-QKW-(SEQ ID NO: 107) (hereinafter referred to as BCY7632);

[0787] Ac-QKW-(SEQ ID NO: 108) (hereinafter referred to as BCY7639);

[0788] Ac-QKW-(SEQ ID NO: 109) (hereinafter referred to as BCY7640);

[0789] Ac-QKW-(SEQ ID NO: 110) (hereinafter referred to as BCY7643);

[0790] Ac-QKW-(SEQ ID NO: 153) (hereinafter referred to as BCY7998);

[0791] Ac-QKW-(SEQ ID NO: 154) (hereinafter referred to as BCY8000);

[0792] A-(SEQ ID NO:35)-A (referred to herein as 80-11-00 or BCY471);

[0793] A-(SEQ ID NO:36)-A (referred to herein as 80-11-01 or BCY472);

[0794] A-(SEQ ID NO:37)-SRF (referred to herein as 80-11-08-T01 or BCY3406);

[0795] Ac-(SEQ ID NO:37)-SRF (referred to herein as BCY7393);

[0796] DDA-(SEQ ID NO:45)-A (hereinafter referred to as BCY3386);

[0797] DTA-(SEQ ID NO:46)-A (hereinafter referred to as BCY3388);

[0798] DSE-(SEQ ID NO:47)-A (hereinafter referred to as BCY3389);

[0799] HDA-(SEQ ID NO:48)-A (hereinafter referred to as BCY3390);

[0800] MDT-(SEQ ID NO:49)-A (hereinafter referred to as BCY3391);

[0801] DPG-(SEQ ID NO: 50)-A (hereinafter referred to as BCY3392);

[0802] HDS-(SEQ ID NO:51)-A (hereinafter referred to as BCY3393);

[0803] (DH)DS-(SEQ ID NO:51)-A (hereinafter referred to as BCY7272);

[0804] A-(SEQ ID NO: 52)-TDK (hereinafter referred to as BCY3394);

[0805] A-(SEQ ID NO: 53)-LKD (hereinafter referred to as BCY3395);

[0806] A-(SEQ ID NO: 54)-TTA (hereinafter referred to as BCY3396);

[0807] A-(SEQ ID NO: 55)-QME (hereinafter referred to as BCY3397);

[0808] A-(SEQ ID NO: 56)-LSE (hereinafter referred to as BCY3398);

[0809] A-(SEQ ID NO: 57)-STD (hereinafter referred to as BCY3399);

[0810] A-(SEQ ID NO: 59)-TNK (hereinafter referred to as BCY7265);

[0811] Ac-(SEQ ID NO:59) (hereinafter referred to as BCY7660);

[0812] A-(SEQ ID NO:60)-TNK (hereinafter referred to as BCY7266);

[0813] Ac-(SEQ ID NO:60) (hereinafter referred to as BCY7616);

[0814] HDS-(SEQ ID NO:61)-A (hereinafter referred to as BCY7273);

[0815] HDS-(SEQ ID NO: 62)-A (hereinafter referred to as BCY7274);

[0816] HDS-(SEQ ID NO: 63)-A (hereinafter referred to as BCY7275);

[0817] HDS-(SEQ ID NO: 64)-A (hereinafter referred to as BCY7276);

[0818] A-(SEQ ID NO:66)-TNK (hereinafter referred to as BCY7349);

[0819] A-(SEQ ID NO: 67)-TNK (hereinafter referred to as BCY7350);

[0820] Ac-(SEQ ID NO:67) (hereinafter referred to as BCY7538);

[0821] A-(SEQ ID NO: 68)-TNK (hereinafter referred to as BCY7359);

[0822] A-(SEQ ID NO:69)-TNK (hereinafter referred to as BCY7360);

[0823] A-(SEQ ID NO:70)-TNK (hereinafter referred to as BCY7361);

[0824] A-(SEQ ID NO:71)-TNK (hereinafter referred to as BCY7365);

[0825] A-(SEQ ID NO:72)-TNK (hereinafter referred to as BCY7370);

[0826] Ac-(SEQ ID NO:73) (hereinafter referred to as BCY7535);

[0827] Ac-(SEQ ID NO:74) (hereinafter referred to as BCY7536);

[0828] Ac-(SEQ ID NO:75) (hereinafter referred to as BCY7541);

[0829] [B-Ala][Sar5]-(SEQ ID NO:76) (hereinafter referred to as BCY7556);

[0830] Ac-[B-Ala][Sar5]-(SEQ ID NO:76) (hereinafter referred to as BCY7558);

[0831] [B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7557);

[0832] Ac-[B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7559);

[0833] Ac-(SEQ ID NO:78) (hereinafter referred to as BCY7580);

[0834] Ac-(SEQ ID NO:79) (hereinafter referred to as BCY7581);

[0835] Ac-(SEQ ID NO:80) (hereinafter referred to as BCY7582);

[0836] Ac-(SEQ ID NO:81) (hereinafter referred to as BCY7584);

[0837] Ac-(SEQ ID NO:82) (hereinafter referred to as BCY7585);

[0838] Ac-(SEQ ID NO:83) (hereinafter referred to as BCY7588);

[0839] Ac-(SEQ ID NO:84) (hereinafter referred to as BCY7589);

[0840] Ac-(SEQ ID NO:85) (hereinafter referred to as BCY7590);

[0841] Ac-(SEQ ID NO:86) (hereinafter referred to as BCY7591);

[0842] Ac-(SEQ ID NO:87) (hereinafter referred to as BCY7592);

[0843] Ac-(SEQ ID NO:88) (hereinafter referred to as BCY7593);

[0844] Ac-(SEQ ID NO:89) (hereinafter referred to as BCY7594);

[0845] Ac-(SEQ ID NO:90) (hereinafter referred to as BCY7595);

[0846] Ac-(SEQ ID NO:91) (hereinafter referred to as BCY7596);

[0847] Ac-(SEQ ID NO:92) (hereinafter referred to as BCY7597);

[0848] Ac-(SEQ ID NO:93) (hereinafter referred to as BCY7598);

[0849] Ac-(SEQ ID NO:94) (hereinafter referred to as BCY7607);

[0850] Ac-(SEQ ID NO:95) (hereinafter referred to as BCY7608);

[0851] Ac-(SEQ ID NO:96) (hereinafter referred to as BCY7611);

[0852] Ac-(SEQ ID NO:97) (hereinafter referred to as BCY7612);

[0853] Ac-(SEQ ID NO:98) (hereinafter referred to as BCY7613);

[0854] Ac-(SEQ ID NO:99) (hereinafter referred to as BCY7614);

[0855] Ac-(SEQ ID NO: 100) (hereinafter referred to as BCY7615);

[0856] Ac-(SEQ ID NO: 101) (hereinafter referred to as BCY7618);

[0857] Ac-(SEQ ID NO: 102) (hereinafter referred to as BCY7620);

[0858] Ac-(SEQ ID NO: 111) (hereinafter referred to as BCY7663);

[0859] Ac-(SEQ ID NO: 112) (hereinafter referred to as BCY7664);

[0860] Ac-(SEQ ID NO: 113) (hereinafter referred to as BCY7667);

[0861] Ac-(SEQ ID NO: 114) (hereinafter referred to as BCY7668);

[0862] Ac-(SEQ ID NO: 115) (hereinafter referred to as BCY7765);

[0863] (SEQ ID NO: 115) (hereinafter referred to as BCY7793);

[0864] (MeO-dPEG12) (SEQ ID NO: 115) (hereinafter referred to as BCY8087);

[0865] (carboxyfluorescein) (SEQ ID NO: 115) (hereinafter referred to as BCY8208);

[0866] (PEG3)(PEG3)(SEQ ID NO:115)(hereinafter referred to as BCY7815);

[0867] (MeO-dPEG12)(PEG3)(PEG3) (SEQ ID NO: 115) (hereinafter referred to as BCY8094);

[0868] Ac-DDD-(SEQ ID NO: 115) (hereinafter referred to as BCY8028);

[0869] Ac-[dD][dD][dD]-(SEQ ID NO: 115) (hereinafter referred to as BCY8029);

[0870] [B-Ala][Sar10]-(SEQ ID NO: 115) (hereinafter referred to as BCY7814);

[0871] Ac-(SEQ ID NO: 116) (hereinafter referred to as BCY7816);

[0872] Ac-(SEQ ID NO: 116)(MeO-dPEG12) linked to D-Lys6 (hereinafter referred to as BCY8084);

[0873] Ac-(SEQ ID NO: 117) (hereinafter referred to as BCY7817);

[0874] Ac-(SEQ ID NO: 118) (hereinafter referred to as BCY7818;

[0875] Ac-(SEQ ID NO: 118)(MeO-dPEG12) is linked to Pro(4NH)1 (hereinafter referred to as BCY8086);

[0876] Ac-(SEQ ID NO: 119) (hereinafter referred to as BCY7819;

[0877] Ac-(SEQ ID NO: 119)(MeO-dPEG12) linked to Lys5 (hereinafter referred to as BCY8088);

[0878] Ac-(SEQ ID NO: 120) (hereinafter referred to as BCY7820;

[0879] Ac-(SEQ ID NO: 120)(MeO-dPEG12) linked to D-Lys3 (hereinafter referred to as BCY8089);

[0880] Ac-(SEQ ID NO: 121) (hereinafter referred to as BCY7821);

[0881] Ac-(SEQ ID NO: 121)(MeO-dPEG12) linked to Lys4 (hereinafter referred to as BCY8090);

[0882] Ac-(SEQ ID NO: 122) (hereinafter referred to as BCY7822);

[0883] Ac-(SEQ ID NO: 122)(MeO-dPEG12) linked to Lys6 (hereinafter referred to as BCY8091);

[0884] Ac-(SEQ ID NO: 123) (hereinafter referred to as BCY7876);

[0885] Ac-(SEQ ID NO: 124) (hereinafter referred to as BCY7877);

[0886] Ac-(SEQ ID NO: 125) (hereinafter referred to as BCY7879);

[0887] Ac-(SEQ ID NO: 126) (hereinafter referred to as BCY7881);

[0888] Ac-(SEQ ID NO: 127) (hereinafter referred to as BCY7883);

[0889] Ac-(SEQ ID NO: 128) (hereinafter referred to as BCY7884);

[0890] Ac-(SEQ ID NO: 129) (hereinafter referred to as BCY7886);

[0891] Ac-(SEQ ID NO: 130) (hereinafter referred to as BCY7887);

[0892] Ac-(SEQ ID NO: 131) (hereinafter referred to as BCY7889);

[0893] Ac-(SEQ ID NO: 132) (hereinafter referred to as BCY7890);

[0894] Ac-(SEQ ID NO: 133) (hereinafter referred to as BCY7891);

[0895] Ac-(SEQ ID NO: 134) (hereinafter referred to as BCY7892);

[0896] Ac-(SEQ ID NO: 135) (hereinafter referred to as BCY7894);

[0897] Ac-(SEQ ID NO: 136) (hereinafter referred to as BCY7895);

[0898] Ac-(SEQ ID NO: 137) (hereinafter referred to as BCY7896);

[0899] Ac-(SEQ ID NO: 138) (hereinafter referred to as BCY7897);

[0900] Ac-(SEQ ID NO: 139) (hereinafter referred to as BCY7902);

[0901] Ac-(SEQ ID NO: 140) (hereinafter referred to as BCY7903);

[0902] Ac-(SEQ ID NO: 141) (hereinafter referred to as BCY7904);

[0903] Ac-(SEQ ID NO: 142) (hereinafter referred to as BCY7906);

[0904] Ac-(SEQ ID NO: 143) (hereinafter referred to as BCY7907);

[0905] Ac-(SEQ ID NO: 144) (hereinafter referred to as BCY7908);

[0906] Ac-(SEQ ID NO: 145) (hereinafter referred to as BCY7911);

[0907] Ac-(SEQ ID NO: 146) (hereinafter referred to as BCY7912);

[0908] Ac-(SEQ ID NO: 147) (hereinafter referred to as BCY7913);

[0909] Ac-(SEQ ID NO: 148) (hereinafter referred to as BCY7914);

[0910] Ac-(SEQ ID NO: 149) (hereinafter referred to as BCY7915);

[0911] Ac-(SEQ ID NO: 150) (hereinafter referred to as BCY7916);

[0912] Ac-(SEQ ID NO: 151) (hereinafter referred to as BCY7973);

[0913] Ac-(SEQ ID NO: 152) (hereinafter referred to as BCY7979);

[0914] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[0915] Ac-(SEQ ID NO: 156) (hereinafter referred to as BCY8031);

[0916] Ac-(SEQ ID NO: 157) (hereinafter referred to as BCY8032);

[0917] Ac-(SEQ ID NO: 158) (hereinafter referred to as BCY8036);

[0918] Ac-(SEQ ID NO: 159) (hereinafter referred to as BCY8037);

[0919] Ac-(SEQ ID NO: 160) (hereinafter referred to as BCY8038);

[0920] Ac-(SEQ ID NO: 161) (hereinafter referred to as BCY8039);

[0921] Ac-(SEQ ID NO: 162) (hereinafter referred to as BCY8040);

[0922] Ac-(SEQ ID NO: 163) (hereinafter referred to as BCY8041);

[0923] Ac-(SEQ ID NO: 164) (hereinafter referred to as BCY8042);

[0924] Ac-(SEQ ID NO: 165) (hereinafter referred to as BCY8042);

[0925] Ac-(SEQ ID NO: 166) (hereinafter referred to as BCY8085);

[0926] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[0927] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[0928] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[0929] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[0930] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[0931] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[0932] (SEQ ID NO: 169) (hereinafter referred to as BCY8116);

[0933] Fluorescein-(SEQ ID NO: 169) (hereinafter referred to as BCY8205);

[0934] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234);

[0935] [PYA][B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8846); Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[0936] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[0937] (SEQ ID NO: 170) (hereinafter referred to as BCY8206);

[0938] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[0939] (SEQ ID NO: 171) (hereinafter referred to as BCY8207);

[0940] [B-Ala][Sar10]-(SEQ ID NO: 171) (hereinafter referred to as BCY8232);

[0941] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[0942] Ac-(SEQ ID NO: 173) (hereinafter referred to as BCY8153);

[0943] Ac-(SEQ ID NO: 174) (hereinafter referred to as BCY8154);

[0944] Ac-(SEQ ID NO: 175) (hereinafter referred to as BCY8157);

[0945] Ac-(SEQ ID NO: 176) (hereinafter referred to as BCY8158);

[0946] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[0947] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[0948] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[0949] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[0950] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[0951] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[0952] [B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8269);

[0953] Ac-(SEQ ID NO: 180) (hereinafter referred to as BCY8174);

[0954] Ac-(SEQ ID NO: 181) (hereinafter referred to as BCY8175);

[0955] Ac-(SEQ ID NO: 182) (hereinafter referred to as BCY8176);

[0956] Ac-(SEQ ID NO: 183) (hereinafter referred to as BCY8177);

[0957] Ac-(SEQ ID NO: 184) (hereinafter referred to as BCY8178);

[0958] Ac-(SEQ ID NO: 185) (hereinafter referred to as BCY8180);

[0959] Ac-(SEQ ID NO: 186) (hereinafter referred to as BCY8181);

[0960] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[0961] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[0962] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[0963] [B-Ala][Sar10]-(SEQ ID NO: 189) (hereinafter referred to as BCY8235);

[0964] Ac-(SEQ ID NO: 190) (hereinafter referred to as BCY8185);

[0965] Ac-(SEQ ID NO: 191) (hereinafter referred to as BCY8186);

[0966] Ac-(SEQ ID NO: 192) (hereinafter referred to as BCY8187);

[0967] Ac-(SEQ ID NO: 193) (hereinafter referred to as BCY8188);

[0968] Ac-(SEQ ID NO: 194) (hereinafter referred to as BCY8189);

[0969] Ac-(SEQ ID NO: 195) (hereinafter referred to as BCY8191);

[0970] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[0971] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[0972] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[0973] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[0974] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[0975] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[0976] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[0977] [B-Ala][Sar10]-(SEQ ID NO: 202) (hereinafter referred to as BCY8231);

[0978] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[0979] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[0980] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280);

[0981] [B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8273);

[0982] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281);

[0983] Ac-(SEQ ID NO: 211) (hereinafter referred to as BCY8831);

[0984] [B-Ala][Sar10]-(SEQ ID NO: 212) (hereinafter referred to as BCY8238);

[0985] (SEQ ID NO: 215) (hereinafter referred to as BCY11415);

[0986] [PYA][B-Ala][Sar10]-(SEQ ID NO: 215) (hereinafter referred to as BCY11942); and (SEQ ID NO: 216) (hereinafter referred to as BCY11414).

[0987] In yet another embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0988] A-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N001 or BCY3385);

[0989] Ac-(SEQ ID NO: 1) (referred to herein as 80-09-02-N008 or BCY7390);

[0990] Ac-(SEQ ID NO: 1)-TNK (referred to herein as 80-09-02-T01-N011 or BCY7391);

[0991] [Ac]QKW-(SEQ ID NO:34) (referred to herein as BCY7392);

[0992] Ac-[B-Ala][Sar5]-(SEQ ID NO: 77) (hereinafter referred to as BCY7559);

[0993] Ac-(SEQ ID NO: 101) (hereinafter referred to as BCY7618);

[0994] Ac-(SEQ ID NO: 115) (hereinafter referred to as BCY7765);

[0995] (SEQ ID NO: 115) (hereinafter referred to as BCY7793);

[0996] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[0997] Ac-(SEQ ID NO: 160) (hereinafter referred to as BCY8038);

[0998] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[0999] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[1000] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[1001] Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[1002] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[1003] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[1004] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[1005] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[1006] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[1007] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[1008] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[1009] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[1010] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[1011] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[1012] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[1013] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[1014] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[1015] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[1016] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[1017] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[1018] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[1019] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[1020] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[1021] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[1022] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[1023] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[1024] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[1025] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[1026] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280); and

[1027] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281).

[1028] The data presented herein in Table 3 demonstrate that the peptide ligands of this embodiment exhibit good levels of binding to human Bindex-4 (<100 nM) as demonstrated by SPR binding data.

[1029] In another embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[1030] Ac-(SEQ ID NO: 155) (hereinafter referred to as BCY8030);

[1031] Ac-[B-Ala][Sar5]-(SEQ ID NO: 167) (hereinafter referred to as BCY8120);

[1032] Ac-[B-Ala][Sar5]-(SEQ ID NO: 168) (hereinafter referred to as BCY8121);

[1033] Ac-[B-Ala][Sar5]-(SEQ ID NO: 169) (hereinafter referred to as BCY8122);

[1034] Ac-[B-Ala][Sar5]-(SEQ ID NO: 170) (hereinafter referred to as BCY8123);

[1035] Ac-(SEQ ID NO: 167) (hereinafter referred to as BCY8124);

[1036] Ac-(SEQ ID NO: 168) (hereinafter referred to as BCY8125);

[1037] Ac-(SEQ ID NO: 169) (hereinafter referred to as BCY8126);

[1038] Ac-(SEQ ID NO: 170) (hereinafter referred to as BCY8127);

[1039] Ac-(SEQ ID NO: 171) (hereinafter referred to as BCY8128);

[1040] Ac-(SEQ ID NO: 172) (hereinafter referred to as BCY8129);

[1041] Ac-(SEQ ID NO: 177) (hereinafter referred to as BCY8161);

[1042] Ac-(SEQ ID NO: 178) (hereinafter referred to as BCY8162);

[1043] Ac-(SEQ ID NO: 179) (hereinafter referred to as BCY8163);

[1044] Ac-(SEQ ID NO: 187) (hereinafter referred to as BCY8182);

[1045] Ac-(SEQ ID NO: 188) (hereinafter referred to as BCY8183);

[1046] Ac-(SEQ ID NO: 189) (hereinafter referred to as BCY8184);

[1047] Ac-(SEQ ID NO: 196) (hereinafter referred to as BCY8192);

[1048] Ac-(SEQ ID NO: 197) (hereinafter referred to as BCY8193);

[1049] Ac-(SEQ ID NO: 198) (hereinafter referred to as BCY8194);

[1050] Ac-(SEQ ID NO: 199) (hereinafter referred to as BCY8211);

[1051] Ac-(SEQ ID NO: 200) (hereinafter referred to as BCY8212);

[1052] Ac-(SEQ ID NO: 201) (hereinafter referred to as BCY8213);

[1053] Ac-(SEQ ID NO: 202) (hereinafter referred to as BCY8214);

[1054] Ac-(SEQ ID NO: 203) (hereinafter referred to as BCY8215);

[1055] Ac-[B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8276);

[1056] Ac-[B-Ala][Sar10]-(SEQ ID NO: 178) (hereinafter referred to as BCY8277);

[1057] Ac-[B-Ala][Sar10]-(SEQ ID NO: 177) (hereinafter referred to as BCY8278);

[1058] Ac-[B-Ala][Sar10]-(SEQ ID NO: 208) (hereinafter referred to as BCY8279);

[1059] Ac-[B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8280); and

[1060] Ac-[B-Ala][Sar10]-(SEQ ID NO: 210) (hereinafter referred to as BCY8281).

[1061] The data presented herein in Table 3 demonstrate that the peptide ligands of this embodiment exhibit excellent levels of binding to human Bindex-4 (<10 nM) as demonstrated by SPR binding data.

[1062] In alternative embodiments, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[1063] [B-Ala][Sar5]-(SEQ ID NO:76) (hereinafter referred to as BCY7556);

[1064] [B-Ala][Sar10]-(SEQ ID NO: 115) (hereinafter referred to as BCY7814);

[1065] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234);

[1066] [B-Ala][Sar10]-(SEQ ID NO: 202) (hereinafter referred to as BCY8231);

[1067] [B-Ala][Sar10]-(SEQ ID NO: 171) (hereinafter referred to as BCY8232);

[1068] [B-Ala][Sar10]-(SEQ ID NO: 189) (hereinafter referred to as BCY8235);

[1069] Ac-(SEQ ID NO: 211) (hereinafter referred to as BCY8831);

[1070] [B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8269); and

[1071] [B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8273).

[1072] The data presented herein in Table 4 demonstrate that the peptide ligands of this embodiment, when conjugated to a cytotoxic agent, exhibit good levels of binding to human nectin-4 (<100 nM), as demonstrated by SPR binding data.

[1073] In yet another embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[1074] [B-Ala][Sar10]-(SEQ ID NO: 169) (hereinafter referred to as BCY8234);

[1075] [B-Ala][Sar10]-(SEQ ID NO: 202) (hereinafter referred to as BCY8231);

[1076] [B-Ala][Sar10]-(SEQ ID NO: 171) (hereinafter referred to as BCY8232);

[1077] [B-Ala][Sar10]-(SEQ ID NO: 189) (hereinafter referred to as BCY8235);

[1078] Ac-(SEQ ID NO: 211) (hereinafter referred to as BCY8831);

[1079] [B-Ala][Sar10]-(SEQ ID NO: 179) (hereinafter referred to as BCY8269); and

[1080] [B-Ala][Sar10]-(SEQ ID NO: 209) (hereinafter referred to as BCY8273).

[1081] The data presented herein in Table 4 demonstrate that the peptide ligands of this embodiment, when conjugated to a cytotoxic agent, exhibit excellent levels of binding to human nectin-4 (<10 nM), as demonstrated by SPR binding data.

[1082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques for molecular biology, genetic, and biochemical methods are described in 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 ed., John Wiley & Sons, Inc., which are incorporated herein by reference.

[1083] Nomenclature

[1084] serial number

[1085] When referring to amino acid residue positions within the peptides of the present invention, cysteine ​​residues are omitted from the numbering because they are invariant (C i 、C ii and C iii ), therefore, the numbering of the amino acid residues in the peptides of the present invention is as follows:

[1086] C i -P1-F2-G3-C ii -M4-K5-N6-W7-S8-W9-P 10 -I 11 -W 12 -C iii (SEQ ID NO: 1).

[1087] For the purpose of this description, it is assumed that all bicyclic peptides are cyclized with 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) to produce a trisubstituted structure. Cyclization with TATA occurs at C i 、C ii and C iii superior.

[1088] Molecular form

[1089] The N- or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence and separated by a hyphen. For example, the N-terminal β-Ala-Sar10-Ala tail would be represented as:

[1090] βAla-Sar10-A-(SEQ ID NO:X).

[1091] Reverse peptide sequence

[1092] It is envisaged that the peptide sequences disclosed herein will also be used in their retro-inverso form, as disclosed in Nair et al. (2003) J Immunol 170(3), 1362-1373. For example, the order is reversed (i.e., the N-terminus becomes the C-terminus and vice versa) and their stereochemistry is inverted (i.e., D-amino acids become L-amino acids and vice versa).

[1093] Peptide ligand

[1094] The peptide ligand referred to herein refers to a peptide that is covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond with the scaffold, and a sequence (referred to as a loop sequence) relative to the reactive groups, which is referred to as a loop sequence because when the peptide is bound to the scaffold, it forms a ring. In the case of the present application, the peptide comprises at least three cysteine ​​residues (referred to herein as C i 、C ii and C iii ), which form at least two rings on the stent.

[1095] Advantages of peptide ligands

[1096] Certain bicyclic peptides of the present invention have a number of advantageous properties that make them suitable for drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. These advantageous properties include:

[1097] - Species cross-reactivity. This is a typical requirement for preclinical efficacy and pharmacokinetic evaluations;

[1098] Protease stability. Bicyclic peptide ligands should ideally 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 lead candidates can be developed in animal models and reliably administered to humans;

[1099] - Ideal solubility profile. This is a function of the ratio of charged hydrophilic residues to hydrophobic residues and intra- / inter-molecular H-bonds, which is important for formulation and absorption purposes;

[1100] - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short exposure in acute disease management settings, or to develop bicyclic peptides with enhanced retention in circulation and therefore preferred for management of more chronic disease states. Other factors driving the desired plasma half-life are the sustained exposure required to achieve maximum therapeutic efficacy versus the toxicology associated with sustained exposure to the agent; and

[1101] - Selectivity. Certain peptide ligands of the present invention exhibit good selectivity over other bindins.

[1102] Pharmaceutically acceptable salts

[1103] It will be understood that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[1104] The salts of the present invention can be synthesized from parent compounds containing a base or acid moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Generally, these salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent or in a mixture of the two.

[1105] Acid addition salts (mono or di) can be formed with various acids (inorganic and organic). Examples of acid addition salts include mono or di salts formed with acids selected from the group consisting of 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, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic 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 (such as hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (such as (+)-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-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.

[1106] A specific group of salts includes 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, propionic acid, butyric acid, malonic acid, glucuronic acid, and lactobionic acid. One specific salt is the hydrochloride. Another specific salt is the acetate.

[1107] If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then a salt may be formed with an organic or inorganic base that generates an appropriate cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li + 、Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R + NH2R2+ 、NHR3 + NR4 + 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(CH3)4 + .

[1108] When the peptides of the present invention contain amine functional groups, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[1109] Modified derivatives

[1110] It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-tolerant amino acid residues; replacement of one or more amino acid residues with alanine, and replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; modification of the peptide backbone length; replacement 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 replacement of amino acids to introduce orthogonal reactivity suitable for functionalization, for example, amino acids carrying azide or alkyne groups allow functionalization with alkyne- or azide-carrying moieties, respectively.

[1111] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and / or a C-terminal modification using suitable carboxyl-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator, or a chromophore.

[1112] 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, during peptide synthesis, the N-terminal cysteine ​​group (which group is referred to herein as C i ) is capped with acetic anhydride or other suitable reagent to produce an N-terminally acetylated molecule. This embodiment provides the advantage of removing potential recognition sites for aminopeptidases and avoiding the possibility of degradation of the bicyclic peptide.

[1113] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of the effector group and maintains the potency of the bicyclic peptide towards its target.

[1114] 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 cysteine ​​group (which group is referred to herein as C iii ) is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment provides the advantage of removing a potential recognition site for carboxypeptidases and reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[1115] 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 can be selected that have isosteric / isoelectronic side chains that are not recognized by degradative proteases and that do not produce any side effects on target efficacy.

[1116] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of adjacent peptide bonds is conformationally and sterically hindered. Specifically, these relate to proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, which are simple derivatives of amino-cyclopropylcarboxylic acids.

[1117] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii )Add a spacer group.

[1118] In one embodiment, the modified derivative comprises the replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues.

[1119] In one embodiment, the derivative of modification comprises one or more charged amino acid residues replaced with one or more hydrophobic amino acid residues. In an alternative embodiment, the derivative of modification comprises one or more hydrophobic amino acid residues replaced with one or more charged amino acid residues. The correct balance of charged amino acid residue and hydrophobic amino acid residue is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residue affects the degree of plasma protein binding, therefore affects the concentration of available free fraction in blood plasma, and charged amino acid residue (specifically arginine) can affect the interaction of peptide and the phospholipid membrane on cell surface. Both combinations can affect the half-life, distribution volume and exposure of peptide drugs, and can be adjusted according to clinical endpoint. In addition, the correct combination and quantity of charged amino acid residue and hydrophobic amino acid residue can reduce the stimulation (if peptide drug is subcutaneously administered) of injection site.

[1120] 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).

[1121] In one embodiment, the modified derivative comprises removal of any amino acid residue and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[1122] It should be noted that each of the modifications mentioned above is used to intentionally improve the efficacy or stability of the peptide. Further efficacy improvements based on modifications can be achieved through the following mechanisms:

[1123] - Incorporation of hydrophobic moieties that exhibit a hydrophobic effect and lead to a reduced off-rate, thereby achieving higher affinity;

[1124] - incorporation of charged groups that exhibit long-range ionic interactions, resulting in faster on-rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[1125] - Incorporating additional constraints into the peptide, for example by properly constraining the amino acid side chains to minimize the entropy loss upon target binding; constraining the torsion angles of the backbone to minimize the entropy loss upon target binding; and introducing additional cyclization into the molecule for the same reason.

[1126] (For reviews see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[1127] Isotopic variation

[1128] The present invention includes all pharmaceutically acceptable (radioactive) isotope-labeled peptide ligands of the present invention, in which one or more atoms are replaced by atoms with the same atomic number, but the atomic mass or mass number is different from the atomic mass or mass number usually found in nature, and the peptide ligands of the present invention are linked to metal chelating groups (referred to as "effectors") that can accommodate the relevant (radioactive) isotopes, and certain functional groups in the peptide ligands of the present invention are covalently replaced with relevant (radioactive) isotopes or isotope-labeled functional groups.

[1129] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, such as 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C. 13 C and 14 C, isotopes of chlorine, such as 36 Cl, isotopes of fluorine, such as 18 F, isotopes of iodine, such as 123 I. 125 I and 131 I, isotopes of nitrogen, such as 13 N and 15 N, isotopes of oxygen, such as 15 O. 17 O and 18 O, isotopes of phosphorus, such as 32 P, isotopes of sulfur, such as 35 S, isotopes of copper, such as 64 Cu, gallium isotopes, such as 67 Ga or 68 Ga, an isotope of yttrium, such as 90 Y, and isotopes of lutetium, such as 177 Lu, and isotopes of bismuth, such as 213 Bi.

[1130] Certain isotope-labeled peptide ligands of the invention, such as those incorporating radioactive isotopes, can be used in drug and / or substrate tissue distribution studies and clinical assessment of the presence and / or absence of the bindin-4 target in diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that 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 labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, fluorescein, photoproteins, and luciferases), and the like. The radioisotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C, is particularly useful for this purpose due to its ease of incorporation and convenient means of detection.

[1131] Using heavier isotopes such as deuterium (i.e. 2 Substitution of H(D)) may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances.

[1132] Using positron-emitting isotopes (such as 11 C. 18 F. 15 O and 13 N) substitutions can be used in positron emission tomography (PET) studies to examine target occupancy.

[1133] 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 non-labeled reagent previously used.

[1134] Molecular scaffold

[1135] 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 comprise an aromatic (ie, unsaturated) carbocyclic or heterocyclic ring system.

[1136] Examples of suitable non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[1137] As described in previous documents, the molecular scaffold can be a small molecule, such as an organic small molecule.

[1138] In one embodiment, the molecular scaffold can be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[1139] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of the polypeptide to form covalent bonds.

[1140] The molecular scaffold may include 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.

[1141] An example of a compound containing an αβ unsaturated carbonyl group is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

[1142] Effectors and functional groups

[1143] According to a further aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[1144] Effectors and / or functional groups can be attached, for example, to the N- and / or C-terminus of a polypeptide, to amino acids within a polypeptide, or to a molecular scaffold.

[1145] Suitable effector groups include antibodies and parts or fragments thereof. For example, the effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, and one or more constant region domains. The effector group can also include the hinge region of an antibody (usually found between the CH1 and CH2 domains of an IgG molecule).

[1146] In a further embodiment of this aspect of the invention, the effector group according to the invention is an Fc region of an IgG molecule. Advantageously, the peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more, two days or more, three days or more, four days or more, five days or more, six days or more, or seven days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more.

[1147] Functional groups typically include binding groups, drugs, reactive groups for linking other entities, functional groups that facilitate the uptake of the macrocyclic peptide into cells, and the like.

[1148] The ability of peptides to penetrate cells will allow the peptides to effectively target intracellular targets. Targets that can be approached by peptides with the ability to penetrate cells include transcription factors, intracellular signaling molecules such as tyrosine kinases and molecules involved in apoptosis pathways. Functional groups that can penetrate cells include peptides or chemical groups that have been added to peptides or molecular scaffolds. Peptides, such as those derived from homeobox proteins such as VP22, HIV-Tat, Drosophila (Antennapedia), for example, as described in Chen and Harrison, Biochemical Society Transactions (2007) Vol. 35, Part 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004) Vol. 57, 9637. The example of short peptides that has been shown to be effective in the translocation of plasma membrane includes 16 amino acid whose penetrating peptides (Derossi et al. (1994) J Biol.Chem. volume 269, page 10444), 18 amino acid whose " model amphipathic peptides " (Oehlke et al. (1998) Biochim Biophys Acts volume 1414, page 127) and HIV TAT protein are rich in arginine region. Non-peptide method includes using the small molecule mimics or SMOC (Okuyama et al. (2007) Nature Methods volume 4, page 153) that can be easily connected to biomolecule. Other chemical strategies that guanidine radicals are added to molecule also enhance cell penetration (Elson-Scwab et al. (2007) J Biol Chem volume 282, page 13585). Small molecular weight molecules (such as steroids) can be added to molecular scaffold to enhance cellular uptake.

[1149] One type of functional group that can be linked to a peptide ligand includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies that bind to proteins that increase the half-life of the peptide ligand in vivo can be used.

[1150] In one embodiment, the peptide ligand-effector group according to the present invention has a tβ half-life selected from 12 hours or longer, 24 hours or longer, 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, 11 days or longer, 12 days or longer, 13 days or longer, 14 days or longer, 15 days or longer, or 20 days or longer. Advantageously, the peptide ligand-effector group or composition according to the present invention will have a tβ half-life in the range of 12 to 60 hours. In a further embodiment, it will have a tβ half-life of one day or longer. In a further embodiment, it will be in the range of 12 to 26 hours.

[1151] In a particular embodiment of the present invention, the functional group is selected from metal chelators, which are suitable for complexing metal radioisotopes of pharmaceutical relevance.

[1152] Possible effector groups also include enzymes such as carboxypeptidase G2 for enzyme / prodrug therapy, where a peptide ligand replaces the antibody in ADEPT.

[1153] In a specific embodiment of the present invention, the functional group is selected from drugs, such as cytotoxic agents used in cancer treatment. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors including acridine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics, including the immunosuppressant actinomycin D (used in kidney transplantation), doxorubicin, epirubicin, bleomycin, calicheamicin, etc.

[1154] In a further embodiment of the invention, the cytotoxic agent is selected from maytansinoids (eg DM1) or monomethyl auristatins (eg MMAE).

[1155] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine with the following structure:

[1156]

[1157] Data are shown herein in Table 4, which demonstrate the effects of peptide ligands conjugated to DM1-containing toxins.

[1158] Monomethyl auristatin E (MMAE) is a synthetic antitumor agent with the following structure:

[1159]

[1160] Data are shown herein in Table 4, which demonstrate the effects of peptide ligands conjugated to MMAE-containing toxins.

[1161] In a further specific embodiment of the present invention, said cytotoxic agent is selected from monomethyl auristatin E (MMAE).

[1162] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide via a cleavable bond (e.g., a disulfide bond or a protease-sensitive bond). In a further embodiment, the groups adjacent to the disulfide bond are modified to control the hindrance of the disulfide bond and thereby control the rate of cleavage and the concomitant release of the cytotoxic agent.

[1163] Published work has identified the potential to alter the sensitivity of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). Greater steric hindrance reduces the rate of reduction by intracellular glutathione and extracellular (systemic) reducing agents, thereby reducing the ease of toxin release inside and outside the cell. Therefore, the optimal balance between disulfide stability in the circulation (which minimizes adverse side effects of the toxin) and efficient release into the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully selecting the degree of hindrance on either side of the disulfide bond.

[1164] The blockage on either side of the disulfide bond is modulated by introducing one or more methyl groups on the targeting entity (here the bicyclic peptide) or toxin side of the molecular construct.

[1165] In one embodiment, the cytotoxic agent and linker are selected from any combination of those cytotoxic agents and linkers described in WO 2016 / 067035 (which cytotoxic agents and linkers are incorporated herein by reference).

[1166] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises one or more amino acid residues. Examples of amino acid residues that are suitable as suitable linkers include Ala, Cit, Lys, Trp and Val.

[1167] In one embodiment, the cytotoxic agent is selected from MMAE, and the drug conjugate further comprises a linker selected from: -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABC represents p-aminobenzylcarbamate. Full details of linkers containing cyclobutyl can be found in Wei et al. (2018) J. Med. Chem. 61, 989-1000. In a further embodiment, the cytotoxic agent is selected from MMAE, and the linker is -PABC-Cit-Val-glutaryl-.

[1168] In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate further comprises a linker which is -SPDB-(SO3H)-, wherein SPDB stands for N-succinimidyl 3-(2-pyridyldithio)propionate.

[1169] In one embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY7556 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY7683. The data presented herein are shown in Table 4, which demonstrates excellent binding of BCY7683 to human bindin-4 in an SPR binding assay. This BDC also demonstrates good anti-tumor activity in a non-small cell lung cancer model, as shown in Example 1.

[1170] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY7814 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY7825. The data shown in this article are shown in Table 4, which demonstrates the excellent binding of BCY7825 to human binding protein-4 in an SPR binding assay. This BDC also demonstrates good anti-tumor activity in a non-small cell lung cancer model (as shown in Example 1) and in a bladder cancer model (as shown in Example 2).

[1171] In an alternative embodiment, the cytotoxic agent is DM1, the bicyclic peptide is selected from BCY7814 as defined herein, and the linker is selected from -SPDB-(SO3H)-. This BDC is referred to herein as BCY7826. The data presented herein are shown in Table 4, which demonstrate excellent binding of BCY7826 to human ligase-4 in an SPR binding assay. This BDC also demonstrates good antitumor activity in a non-small cell lung cancer model (as shown in Example 1).

[1172] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8245 and is schematically represented as:

[1173]

[1174] And a more detailed way is as follows:

[1175]

[1176] As shown in Table 4, the data presented herein demonstrate that BCY8245 binds very well to human bindin-4 in an SPR binding assay. This BDC also demonstrated good anti-tumor activity in a non-small cell lung cancer model as shown in Example 1, a bladder cancer model as shown in Example 2, a pancreatic cancer model as shown in Example 3, and a breast cancer model as shown in Example 4.

[1177] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8231 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8253. As shown in Table 4, the data provided herein demonstrate that BCY8253 binds very well to human bindin-4 in an SPR binding assay. This BDC also demonstrated good anti-tumor activity in a non-small cell lung cancer model as shown in Example 1, a bladder cancer model as shown in Example 2, a pancreatic cancer model as shown in Example 3, and a breast cancer model as shown in Example 4.

[1178] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8232 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8254. As shown in Table 4, the data provided herein demonstrate that BCY8254 binds very well to human Bindex-4 in an SPR binding assay. This BDC also demonstrated good anti-tumor activity in the non-small cell lung cancer model described in Example 1 and the bladder cancer model described in Example 2.

[1179] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8235 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8255. As shown in Table 4, the data provided herein demonstrate that BCY8255 binds very well to human bindin-4 in an SPR binding assay. This BDC also demonstrated good anti-tumor activity in a non-small cell lung cancer model as shown in Example 1, a bladder cancer model as shown in Example 2, a pancreatic cancer model as shown in Example 3, and a breast cancer model as shown in Example 4.

[1180] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 as defined herein, and the linker is selected from -PABC-cyclobutyl-(B-Ala)-. This BDC is referred to herein as BCY8549. As shown in Table 4, the data provided herein demonstrate that BCY8549 binds very well to human nectin-4 in an SPR binding assay.

[1181] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8831 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-, and the linker-cytotoxic agent is attached to the bicyclic peptide at the Lys3 position. This BDC is referred to herein as BCY8550. As shown in Table 4, the data provided herein demonstrate that BCY8550 binds very well to human nectin-4 in an SPR binding assay.

[1182] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8269 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8783. As shown in Table 4, the data provided herein demonstrate that BCY8783 binds very well to human Bindex-4 in an SPR binding assay. This BDC also effectively causes tumor regression in a non-small cell lung cancer model as shown in Example 5.

[1183] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8273 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is referred to herein as BCY8784. As shown in Table 4, the data provided herein demonstrate that BCY8784 binds very well to human Bindex-4 in an SPR binding assay. As shown in Example 5, this BDC also effectively causes tumor regression in a non-small cell lung cancer model.

[1184] In another embodiment, the bicyclic drug conjugate is selected from any one of the following: BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, BCY8255, BCY8549, BCY8550, BCY8783, and BCY8784. In another embodiment, the bicyclic drug conjugate is selected from any one of the following: BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, BCY8255, BCY8783, and BCY8784. In yet another embodiment, the bicyclic drug conjugate is BCY8245. As shown in the data described herein, the drug conjugate BCY8245 exhibits superior dose-dependent anti-tumor activity.

[1185] In one embodiment, the drug conjugate is not BCY8245 and / or BCY8549.

[1186] synthesis

[1187] The peptides of the present invention can be synthesized by standard techniques and then reacted with a molecular scaffold in vitro. When this is done, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be implemented using conventional chemical methods disclosed in, for example, Timmerman et al. (supra).

[1188] Therefore, the present invention also relates to the preparation of a selected polypeptide or conjugate as described herein, wherein the preparation comprises the optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide / conjugate prepared by chemical synthesis.

[1189] When preparing conjugates or complexes, amino acid residues in the polypeptide of interest may optionally be substituted.

[1190] The peptide may also be extended to incorporate, for example, another loop, thereby introducing multispecificity.

[1191] To extend the peptide, standard solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs), simply at its N-terminus or C-terminus or chemical extension within the loop. Standard (bio) conjugation techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, addition can be performed by fragment condensation or native chemical ligation (e.g., as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779)), or by enzymes, for example, using subtiligase as described in (Chang et al. Proc Natl Acad Sci US A. 1994 Dec 20; 91(26): 12544-8 or Hikari et al. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, pp. 6000-6003).

[1192] Alternatively, the peptide can be extended or modified by further conjugation via disulfide bonds. This has the additional advantage of allowing the first and second peptides to be separated from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; an additional cysteine ​​or thiol can then be attached to the N- or C-terminus of the first peptide so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[1193] Similar techniques are also applicable to the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[1194] In addition, the addition of other functional groups or effector groups can be accomplished in the same manner using appropriate chemical methods at the N- or C-terminus or through side chain coupling. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

[1195] Pharmaceutical composition

[1196] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising the peptide ligand or drug conjugate as defined herein and one or more pharmaceutically acceptable excipients.

[1197] Generally, the peptide ligands of the present invention will be used in purified form together with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / water solutions, emulsions or suspensions, including physiological saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's reagent. If it is necessary to maintain the polypeptide complex in suspension, suitable physiologically acceptable adjuvants can be selected from thickening agents such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, and alginates.

[1198] 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 ed.).

[1199] The peptide ligands of the present invention can be used as compositions for administration alone or in combination with other agents. These can include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin, or cisplatin, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxins or other agents combined with the protein ligands of the present invention, or even combinations of polypeptides selected according to the present invention with different specificities, such as polypeptides selected using different target ligands, whether or not combined prior to administration.

[1200] The routes of administration of the pharmaceutical compositions according to the present invention may be those generally known to those of ordinary skill in the art. For treatment, the peptide ligands of the present invention may be administered to any patient according to standard techniques. Administration may be by any appropriate mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary routes, or, where appropriate, by direct catheter infusion. Preferably, the pharmaceutical compositions according to the present invention are administered by inhalation. The dosage and frequency of administration will depend on the patient's age, sex, and condition, concurrent administration of other medications, contraindications, and other parameters to be considered by the clinician.

[1201] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and lyophilization and reconstitution techniques known in the art can be employed. It will be understood by those skilled in the art that lyophilization and reconstitution may result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[1202] Compositions containing the peptide ligands of the present invention or cocktails thereof can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, a sufficient amount to achieve at least partial inhibition, suppression, modulation, 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 and the general state of the patient's own immune system, but will typically range from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being more common. 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.

[1203] Compositions comprising the peptide ligands according to the present invention can be used to help alter, inactivate, kill or remove selected target cell populations in mammals in both preventive and therapeutic settings. In addition, the peptide ligands described herein can be selectively used to kill, deplete or otherwise effectively remove target cell populations from heterogeneous collections of cells in vitro or in vitro. Blood from a mammal can be combined with the peptide ligands of selection in vitro, whereby undesirable cells are killed or otherwise removed from the blood and returned to the mammal according to standard techniques.

[1204] Co-administration with one or more other therapeutic agents

[1205] Depending on the specific condition, or disease, to be treated, other therapeutic agents that are typically administered to treat that condition may also be present in the compositions of the present invention. Thus, in one embodiment, the pharmaceutical composition additionally comprises one or more therapeutic agents. As used herein, other therapeutic agents that are typically administered to treat a specific disease, or condition are known as "appropriate for the disease, or condition being treated."

[1206] In some embodiments, the present invention provides a method of treating a disclosed disease or condition, comprising administering to a patient in need thereof an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and simultaneously or sequentially administering an effective amount of one or more other therapeutic agents, such as those described herein. In some embodiments, the method comprises co-administering one other therapeutic agent. In some embodiments, the method comprises co-administering two other therapeutic agents. In some embodiments, the combination of the disclosed compounds and the other one or more therapeutic agents acts synergistically.

[1207] The compounds of the present invention may also be used in combination with known treatments (eg, administration of hormones or radiation). In certain embodiments, provided compounds are useful as radiosensitizers, particularly for treating tumors that are poorly sensitive to radiation therapy.

[1208] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds, and possible combination therapies take the form of fixed combinations, or staggered administration or independent administration of the compounds of the present invention and one or more other therapeutic compounds, or combined administration of fixed combinations and one or more other therapeutic compounds. The compounds of the present invention may also be administered additionally or in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof, particularly for the treatment of tumors. As described above, in the context of other treatment strategies, long-term therapy and adjuvant therapy are equally likely. Other possible treatments are therapies that maintain the patient's status after tumor regression, or even chemopreventive therapy, for example, in patients at risk.

[1209] As part of a multiple dose regimen, one or more other therapeutic agents can be used separately from the compounds of the present invention or compositions. Alternatively, one or more other therapeutic agents can be part of a single dosage form, mixed in a single composition with the compounds of this invention. If used in a multiple dose regimen, one or more other therapeutic agents and the compounds of the present invention or compositions can be used simultaneously, sequentially or within a period of time at intervals of one another, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, one or more other therapeutic agents and the compounds of the present invention or compositions are used in a multiple dose regimen greater than 24 hours apart.

[1210] As used herein, the terms "combination," "combined," and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the present invention. For example, a compound of the present invention can be administered with one or more other therapeutic agents simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Thus, the present invention provides a single unit dosage form comprising a compound of the present invention, one or more other therapeutic agents, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[1211] The amount of the compound of the invention and one or more other therapeutic agents (in those compositions comprising other therapeutic agents as described above) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Preferably, the compositions of the invention should be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the compound of the invention can be administered.

[1212] In those compositions comprising one or more additional therapeutic agents, the one or more additional therapeutic agents and the compounds of the present invention may act synergistically. Thus, the amount of the one or more additional therapeutic agents in such compositions may be less than that required as a monotherapy using only that therapeutic agent. In such compositions, the one or more additional therapeutic agents may be administered at a dose of 0.01–1,000 μg / kg body weight / day.

[1213] The amount of one or more other therapeutic agents present in the compositions of the present invention may not exceed the amount typically administered in a composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of one or more other therapeutic agents in the compositions of the present disclosure will range from about 50% to 100% of the amount typically present in a composition comprising the agent as the sole therapeutic active agent. In some embodiments, one or more other therapeutic agents are administered at a dosage of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount typically administered of the agent. As used herein, the term "typically administered" refers to the amount of an FDA-approved therapeutic agent for administration provided according to the FDA label insert.

[1214] The compounds of the present invention, or their pharmaceutical compositions, can also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents have been used to overcome stenosis (restenosis of the blood vessel wall after injury). However, patients using stents or other implantable devices are at risk of developing blood clots or activated platelets. These adverse effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the present invention.

[1215] Exemplary Other Therapeutic Agents

[1216] In some embodiments, the one or more additional therapeutic agents is a poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib ( AstraZeneca; rucaparib ( Clovis Oncology; niraparib ( Tesaro); talazoparib (MDV3800 / BMN 673 / LT00673, Medivation / Pfizer / Biomarin); veliparib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

[1217] In some embodiments, the one or more additional therapeutic agents are histone deacetylase (HDAC) inhibitors. In some embodiments, the HDAC inhibitor is selected from vorinostat ( Merck; romidepsin ( Celgene; panobinostat ( Novartis; belinostat ( Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide ( HBI-8000, Chipscreen Biosciences, China).

[1218] In some embodiments, the one or more additional therapeutic agents is a CDK inhibitor, e.g., a CDK4 / CDK6 inhibitor. In some embodiments, the CDK 4 / 6 inhibitor is selected from palbociclib ( Pfizer; ribociclib ( Novartis); abemaciclib (Ly2835219, Eli Lilly); and trilaciclib (G1T28, G1 Therapeutics).

[1219] In some embodiments, the one or more additional therapeutic agents is a phosphatidylinositol 3-kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is selected from idelalisib ( Gilead), alpelisib (BYL719, Novartis), taselisib (GDC-0032, Genentech / Roche); pictilisib (GDC-0941, Genentech / Roche); copanlisib (BAY806946, Bayer); duvelisib (formerly IPI-145, Infinity Pharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202 (formerly RP5230, TG Therapeutics).

[1220] In some embodiments, the one or more additional therapeutic agents are platinum-based therapeutic agents, also known as platins. Platinum primarily causes DNA crosslinking in rapidly multiplying cells (e.g., cancer cells), thereby inhibiting DNA repair and / or DNA synthesis. In some embodiments, the platinum-based therapy is selected from cisplatin ( Bristol-Myers Squibb; carboplatin (( Bristol-Myers Squibb; and Teva; Pfizer); oxaliplatin ( Sanofi-Aventis; nedaplatin ( Shionogi), picoplatin (Poniard Pharmaceuticals); and satraplatin (JM-216, Agennix).

[1221] In some embodiments, the one or more additional therapeutic agents is a taxane compound, which causes disruption of microtubules, which are essential for cell division. In some embodiments, the taxane compound is selected from paclitaxel ( Bristol-Myers Squibb, docetaxel ( Sanofi-Aventis; Sun Pharmaceutical); albumin-bound paclitaxel ( Abraxis / Celgene; Cabazitaxel ( Sanofi-Aventis) and SID530 (SK Chemicals, Co.) (NCT00931008).

[1222] In some embodiments, the one or more additional therapeutic agents are nucleoside inhibitors, or therapeutic agents that interfere with normal DNA synthesis, protein synthesis, cell replication, or will inhibit rapidly proliferating cells.

[1223] In some embodiments, the nucleoside inhibitor is selected from trabectedin (a guanidine alkylating agent, Janssen Oncology), mechlorethamine (alkylating agent, Aktelion Pharmaceuticals), vincristine ( Eli Lilly; Teva Pharmaceuticals; Talon Therapeutics; temozolomide (a prodrug of the alkylating agent 5-(3-methyltriaza-1-yl)-imidazole-4-carboxamide (MTIC), Merck); cytarabine injection (ara-C, an antimetabolite cytidine analog, Pfizer); lomustine (alkylating agent, Bristol-Myers Squibb; NextSource Biotechnology); azacitidine (a pyrimidine nucleoside analog of cytosine, Celgene; omacetaxine mepesuccinate (ceftazidime axetil) (protein synthesis inhibitor, Teva Pharmaceuticals); and asparaginase from Erwinia chrysanthemi (an enzyme for removing asparagine, Lundbeck; EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based antimitotic agent, Eisai); cabazitaxel (microtubule inhibitor, tubulin-based antimitotic agent, Sanofi-Aventis; capecitabine (thymidylate synthase inhibitor, Genentech; bendamustine (a bifunctional methylethylamine derivative believed to form interstrand DNA crosslinks, Cephalon / Teva); ixabepilone (a semisynthetic analog of epothilone B, a microtubule inhibitor, and a tubulin-based antimitotic agent, Bristol-Myers Squibb; nelarabine (prodrug of deoxyguanosine analog, nucleoside metabolism inhibitor, Novartis); clorafabine (a prodrug of a ribonucleotide reductase inhibitor, a competitive inhibitor of deoxycytidine, Sanofi-Aventis); and trifluridine and tipiracil (thymidine-based nucleoside analogs and thymidine phosphorylase inhibitors, Taiho Oncology).

[1224] In some embodiments, the one or more additional therapeutic agents are kinase inhibitors or VEGF-R antagonists. Approved VEGF inhibitors and kinase inhibitors that can be used in the present invention include: bevacizumab ( Genentech / Roche), an anti-VEGF monoclonal antibody; ramucirumab ( Eli Lilly, an anti-VEGFR-2 antibody and ziv-aflibercept, also known as VEGF Trap ( Regeneron / Sanofi). VEGFR inhibitors, such as regorafenib ( Bayer; vandetanib ( AstraZeneca; axitinib ( Pfizer; lenvatinib ( Eisai); Raf inhibitors, such as sorafenib ( Bayer AG and Onyx); dabrafenib ( Novartis; and vemurafenib ( Genentech / Roche); MEK inhibitors, such as cobimetanib ( Exelexis / Genentech / Roche; trametinib ( Novartis) Bcr-Abl tyrosine kinase inhibitors, such as imatinib ( Novartis; nilotinib ( Novartis; dasatinib ( BristolMyersSquibb; bosutinib ( Pfizer; and ponatinib ( Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such as gefitinib ( AstraZeneca; erlotinib ( Genentech / Roche / Astellas; lapatinib ( Novartis; afatinib ( Boehringer Ingelheim; osimertinib (targeting activated EGFR, AstraZeneca; and brigatinib ( Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as Cabozantinib ( Exelexis); and multikinase inhibitors, such as sunitinib ( Pfizer; pazopanib ( Novartis); ALK inhibitors, such as crizotinib ( Pfizer; ceritinib ( Novartis; and alectinib ( Genentech / Roche); Bruton's tyrosine kinase inhibitors, such as ibrutinib ( Pharmacyclics / Janssen); and Flt3 receptor inhibitors, such as midostaurin ( Novartis).

[1225] Other kinase inhibitors and VEGF-R antagonists that are in development and may be used in the present invention include: tivozanib (Aveo Pharmaceuticals); vatalanib (Bayer / Novartis); lucitanib (Clovis Oncology); dovitinib (TKI258, Novartis); Chiauanib (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories); neratinib (HKI-272, Puma Biotechnology); radotinib ( IY5511, Il-Yang Pharmaceuticals, S.Korea); ruxolitinib ( Incyte Corporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo); and motesanib (Amgen / Takeda).

[1226] In some embodiments, the one or more additional therapeutic agents is an mTOR inhibitor, which inhibits cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitor is everolimus ( Novartis; temsirolimus ( Pfizer; and sirolimus ( Pfizer).

[1227] In some embodiments, one or more additional therapeutic agents is a proteasome inhibitor. Approved proteasome inhibitors useful in the present invention include bortezomib ( Takeda and carfilzomib ( Amgen); and ixazomib ( Takeda).

[1228] In some embodiments, the one or more additional therapeutic agents is a growth factor antagonist, such as an antagonist of platelet-derived growth factor (PDGF), or an antagonist of epidermal growth factor (EGF) or an antagonist of its receptor (EGFR). Approved PDGF antagonists that can be used in the present invention include olaratumab ( Eli Lilly). Approved EGFR antagonists that can be used in the present invention include: Cetuximab ( Eli Lilly; necitumumab ( Eli Lilly), panitumumab ( Amgen); and osimertinib (targeting activated EGFR, AstraZeneca).

[1229] In some embodiments, the one or more additional therapeutic agents is an aromatase inhibitor. In some embodiments, the aromatase inhibitor is selected from the group consisting of: exemestane ( Pfizer; anastazole ( AstraZeneca and letrozole ( Novartis).

[1230] In some embodiments, one or more additional therapeutic agents is an antagonist of the hedgehog pathway. Approved hedgehog pathway inhibitors that can be used in the present invention include: sonidegib ( Sun Pharmaceuticals; and vismodegib ( Genentech), both used to treat basal cell carcinoma.

[1231] In some embodiments, the one or more additional therapeutic agents is a folate inhibitor. Approved folate inhibitors for use in the present invention include pemetrexed ( Eli Lilly).

[1232] In some embodiments, the one or more additional therapeutic agents is a CC chemokine receptor 4 (CCR4) inhibitor. CCR4 inhibitors under investigation for use in the present invention include mogamulizumab ( Kyowa Hakko Kirin, Japan).

[1233] In some embodiments, the one or more additional therapeutic agents are isocitrate dehydrogenase (IDH) inhibitors. Investigational IDH inhibitors that may be used in the present invention include: AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); IDH305 (Novartis, NCT02987010).

[1234] In some embodiments, the one or more other therapeutic agents are arginase inhibitors. Arginase inhibitors under investigation that can be used in the present invention include: AEB1102 (PEGylated recombinant arginase, Aeglea Biotherapeutics), which is being studied in Phase 1 clinical trials for acute myeloid leukemia and myelodysplastic syndrome (NCT02732184) and solid tumors (NCT02561234); and CB-1158 (Calithera Biosciences).

[1235] In some embodiments, the one or more additional therapeutic agents is a glutaminase inhibitor. Investigational glutaminase inhibitors that may be used in the present invention include CB-839 (Calithera Biosciences).

[1236] In some embodiments, the one or more other therapeutic agents are antibodies that bind to tumor antigens (i.e., proteins expressed on the cell surface of tumor cells). Approved antibodies that bind to tumor antigens that can be used in the present invention include: rituximab ( Genentech / Biogen Idec; ofatumumab (anti-CD20, GlaxoSmithKline; obinutuzumab (anti-CD20, Genentech), ibritumomab (anti-CD20 and Yttrium-90, Spectrum Pharmaceuticals); daratumumab (anti-CD38, Janssen Biotech), dinutuximab (anti-glycolipid GD2, United Therapeutics; trastuzumab (anti-HER2, Genentech; ado-trastuzumabemtansine (anti-HER2, fused to ado-trastuzumab, Genentech); and pertuzumab (anti-HER2, Genentech); and brentuximab vedotin (anti-CD-30 drug conjugate, Seattle Genetics).

[1237] In some embodiments, one or more additional therapeutic agents is a topoisomerase inhibitor. Approved topoisomerase inhibitors useful in the present invention include: irinotecan ( Merrimack Pharmaceuticals); and topotecan ( GlaxoSmithKline). Investigational topoisomerase inhibitors that may be used in the present invention include pixantrone ( CTI Biopharma).

[1238] In some embodiments, one or more additional therapeutic agents is an inhibitor of an anti-apoptotic protein, such as BCL-2. Approved anti-apoptotic agents that can be used in the present invention include: venetoclax ( AbbVie / Genentech; and blinatumomab ( Other therapeutic agents targeting apoptotic proteins that have been clinically tested and can be used in the present invention include navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

[1239] In some embodiments, one or more additional therapeutic agents is an androgen receptor inhibitor. Approved androgen receptor inhibitors that can be used in the present invention include enzalutamide ( Astellas / Medivation; approved androgen synthesis inhibitors include abiraterone ( Centocor / Ortho); approved gonadotropin-releasing hormone (GnRH) receptor antagonists (degaralix, Ferring Pharmaceuticals).

[1240] In some embodiments, the one or more other therapeutic agents are selective estrogen receptor modulators (SERMs), which interfere with the synthesis or activity of estrogen. Approved SERMs that can be used in the present invention include raloxifene ( Eli Lilly).

[1241] In some embodiments, the one or more additional therapeutic agents is an inhibitor of bone resorption. Approved therapeutics that inhibit bone resorption are Denosumab ( Amgen), an antibody that binds to RANKL and blocks binding to its receptor RANK present on the surface of osteoclasts, their precursors, and osteoclast-like giant cells, which mediates bone pathology in solid tumors with bone metastases. Other approved therapeutic agents that inhibit bone resorption include bisphosphonates, such as zoledronic acid ( Novartis).

[1242] In some embodiments, one or more other therapeutic agents are inhibitors of the interaction between the two main p53 inhibitory proteins MDMX and MDM2. Inhibitors of p53 inhibitory proteins under investigation that can be used in the present invention include ALRN-6924 (Aileron), a stapled peptide that binds to and disrupts the interaction of MDMX and MDM2 with p53. ALRN-6924 is currently being evaluated in clinical trials to treat AML, advanced myelodysplastic syndrome (MDS), and peripheral T-cell lymphoma (PTCL) (NCT02909972; NCT02264613).

[1243] In some embodiments, the one or more additional therapeutic agents is an inhibitor of transforming growth factor-β (TGF-β or TGFβ). Investigational inhibitors of TGF-β proteins that can be used in the present invention include NIS793 (Novartis), an anti-TGF-β antibody that is being clinically tested for the treatment of various cancers, including breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer (NCT 02947165). In some embodiments, the inhibitor of TGF-β protein is fresolimumab (GC1008; Sanofi-Genzyme), which is being studied for melanoma (NCT00923169); renal cell carcinoma ((NCT00356460); and non-small cell lung cancer (NCT02581787). Additionally, in some embodiments, the additional therapeutic agent is a TGF-β trap, e.g., as described in Connolly et al. (2012) Int'l J. Biological Sciences 8:964-978. One therapeutic compound currently in clinical trials for the treatment of solid tumors is M7824 (Merck KgaA (formerly known as MSB0011459X), a bispecific anti-PD-L1 / TGFβ trap compound (NCT02699515); and M7824 (NCT02517398). M7824 consists of a fully human IgG1 anti-PD-L1 antibody fused to the extracellular domain of human TGF-β receptor II, which functions as a TGFβ "trap."

[1244] In some embodiments, the one or more additional therapeutic agents are selected from glembatumumab vedotin-monomethyl auristatin E (MMAE) (Celldex), an anti-glycoprotein NMB (gpNMB) antibody (CR011) linked to the cytotoxic MMAE. gpNMB is a protein overexpressed by various tumors and associated with the metastatic ability of various cancer cells.

[1245] In some embodiments, the one or more additional therapeutic agents are anti-proliferative compounds. Such anti-proliferative compounds include, but are not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; anti-tumor antimetabolites; platinum compounds; compounds that target / reduce the activity of protein or lipid kinases and other anti-angiogenic compounds; compounds that target, reduce or inhibit the activity of protein or lipid phosphatases; gonadotropin-releasing hormone agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; anti-proliferative antibodies; heparanase inhibitors; inhibitors of oncogenic isoforms of Ras; telomerase inhibitors; proteasome inhibitors; compounds used to treat hematological malignancies; compounds that target, reduce or inhibit the activity of Flt-3; Hsp90 inhibitors, such as those from Conforma Therapeutics' 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010; and temozolomide Kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine / chlorpromazine from CombinatoRx; MEK inhibitors, such as ARRY142886 from Array BioPharma, AZd6244 from AstraZeneca, PD181461 from Pfizer, and leucovorin.

[1246] As used herein, the term "aromatase inhibitor" relates to compounds that inhibit the production of estrogens, for example, by converting the substrates androstenedione and testosterone into estrone and estradiol, respectively. The term includes, but is not limited to, steroids, particularly adamantane, exemestane and formestane, and particularly non-steroidal, particularly aminoglutamine, rosiglitamide, pyridine glutamate, triclosan, testolactone, ketoconazole, vorozole, faldrozole, anastrozole and letrozole. Exemestane is sold under the trade name Aromasin TM Formestane is sold under the trade name Lentaron TMFadrozole is sold under the trade name Afema TM Anastrozole is sold under the trade name Arimidex. TM Letrozole is sold under the trade name Femara. TM or Femar TM Aminoglutamine is sold under the trade name Orimeten. TM The combination of the invention comprising a chemotherapeutic agent which is an aromatase inhibitor is particularly useful in the treatment of hormone receptor positive tumors, such as breast tumors.

[1247] As used herein, the term "antiestrogen" refers to compounds that antagonize the effects of estrogens at the estrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen is sold under the trade name Nolvadex. TM Raloxifene hydrochloride is sold under the trade name Evista TM Fulvestrant is sold under the trade name Faslodex. TM Combinations of the invention comprising a chemotherapeutic agent that is an antiestrogen are particularly useful in the treatment of estrogen receptor positive tumors, such as breast tumors.

[1248] As used herein, the term "antiandrogen" refers to any substance that is capable of inhibiting the biological effects of androgens, including but not limited to bicalutamide (Casodex TM The term "gonadotropin-releasing agonist" as used herein includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin is available under the trade name Zoladex. TM Application.

[1249] The term "topoisomerase I inhibitors" as used herein includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, for example, in the form as it is marketed, for example, at Camptosar. TM Topotecan is sold under the trade name Hycamptin TM Sale.

[1250] As used herein, the term "topoisomerase II inhibitors" includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations such as Caelyx TM ), daunorubicin, epirubicin, idarubicin and neorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophillotoxines etoposide and teniposide. Etoposide is sold under the trade name Etopophos TM Teniposide is sold under the trade name VM 26-Bristol. Doxorubicin is sold under the trade name Acriblastin TM or Adriamycin TM Epirubicin is sold under the trade name Farmorubicin TM Idarubicin is sold under the trade name Zavedos TM Mitoxantrone is sold under the trade name Novantron.

[1251] The term "microtubule active agent" refers to microtubule stabilizing, microtubule destabilizing compounds and tubulin polymerization inhibitors, including but not limited to: taxanes, such as paclitaxel and docetaxel; vinca alkaloids, such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolides; colchicine and epothilones and their derivatives. Paclitaxel is sold under the trade name Taxol TM Docetaxel is sold under the trade name Taxotere TM Vinblastine sulfate is sold under the trade name Vinblastin RP TM Vincristine sulfate is sold under the trade name Farmistin TM Sale.

[1252] The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is sold under the trade name Cyclostin TM Ifosfamide is sold under the trade name Holoxan TM Sale.

[1253] The term "histone deacetylase inhibitor" or "HDAC inhibitor" relates to compounds that inhibit histone deacetylase and have antiproliferative activity. This includes, but is not limited to, suberoylanilide hydroxamic acid (SAHA).

[1254] The term "antineoplastic antimetabolite" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folic acid antagonists such as pemetrexed. Capecitabine is marketed under the trade name Xeloda TM Gemcitabine is sold under the trade name Gemzar TM Sale.

[1255] The term "platinum compound" as used herein includes, but is not limited to, carboplatin, cisplatin, cisplatin and oxaliplatin. Carboplatin is available, for example, under the trademark Carboplat TM Oxaliplatin can be administered in the form marketed as such. TM The following sales form applies.

[1256] As used herein, the term "compounds that target / decrease the activity of a protein or lipid kinase; or a protein or lipid phosphatase; or other anti-angiogenic compounds" includes, but is not limited to, protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, such as: a) compounds that target, decrease or inhibit the activity of platelet-derived growth factor receptor (PDGFR), such as compounds that target, decrease or inhibit the activity of PDGFR, in particular compounds that inhibit PDGF receptors, such as N-phenyl-2-pyrimidinyl-amine derivatives, such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target, decrease or inhibit the activity of fibroblast growth factor receptor (FGFR); c) compounds that target, decrease or inhibit the activity of insulin-like growth factor receptor I (IGF-IR), such as compounds that target, decrease or inhibit the activity of IGF-IR, in particular compounds that inhibit the kinase activity of IGF-I receptor, or antibodies that target the extracellular domain of the IGF-1 receptor or its growth factors; d) compounds that target, decrease or inhibit the activity of the Trk receptor tyrosine kinase family , or ephrin B4 inhibitors; e) compounds that target, reduce or inhibit the activity of the AxI receptor tyrosine kinase family; f) compounds that target, reduce or inhibit the activity of the Ret receptor tyrosine kinase; g) compounds that target, reduce or inhibit the activity of the Kit / SCFR receptor tyrosine kinase, such as imatinib; h) compounds that target, reduce or inhibit the activity of the C-kit receptor tyrosine kinase, which is part of the PDGFR family, such as compounds that target, reduce or inhibit the c-Kit receptor tyrosine kinase. Compounds that inhibit the activity of the c-Abl family of kinases, in particular compounds that inhibit the c-Kit receptor, such as imatinib; i) compounds that target, reduce or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinase), and mutants, for example, compounds that target, reduce or inhibit the activity of c-Abl family members and their gene fusion products, such as N-phenyl-2-pyrimidinyl-amine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from Parke Davis; or dasatinib (BMS-354825); j) compounds that target, decrease or inhibit the activity of members of the Raf family of protein kinase C (PKC) and serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families, and / or members of the cyclin-dependent kinase family (CDK), including staurosporine derivatives such as midostaurin;Examples of other compounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531 / LY379196; Isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); k) compounds that target, decrease or inhibit the activity of protein tyrosine kinase inhibitors, such as compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, including imatinib mesylate (Gleevec; TM ), or tyrosine phosphorylation inhibitors (tyrphostin), such as Tyrphostin A23 / RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44(+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adenosine deaminase phosphorylation inhibitors (adaphostin) (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester; NSC 680410, adenosine deaminase phosphorylation inhibitors); 1) compounds that target, reduce or inhibit the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR1, ErbB2, ErbB3, ErbB4 as homodimers or heterodimers), or mutants thereof, such as compounds that target, reduce or inhibit the activity of the epidermal growth factor receptor family, specifically compounds, proteins or antibodies that inhibit EGF receptor tyrosine kinase family members, such as EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or bind to EGF-related ligands, CP358774, ZD 1839, ZM 105180; trastuzumab (Herceptin TM ), Cetuximab (Erbitux TM), Iressa, Tarceva, OSI-774, Cl-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrole-[2,3-d]pyrimidine derivatives; m) compounds that target, decrease or inhibit c-Met receptor activity, such as compounds that target, decrease or inhibit c-Met activity, especially compounds that inhibit c-Met receptor kinase activity, or antibodies targeting the extracellular domain of c-Met or antibodies that bind to HGF, n) compounds that target, reduce or inhibit the kinase activity of one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-1 o) compounds that target, decrease or inhibit PI3 kinase (PI3K) kinase activity, including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, and dabrafenib. p) compounds that target, reduce or inhibit the signaling effects of the hedgehog (Hh) or smoothened receptor (SMO) pathways, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib and IPI-926 (saridegib).

[1257] As used herein, the term "PI3K inhibitor" includes, but is not limited to, compounds that have inhibitory activity against one or more enzymes in the phosphatidylinositol 3-kinase family, including, but not limited to, PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87. Examples of PI3K inhibitors useful in the present invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idarisib.

[1258] As used herein, the term "Bcl-2 inhibitor" includes, but is not limited to, compounds that have inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including, but not limited to, ABT-199, ABT-731, ABT-737, apogossypol, Ascenta's pan-Bcl-2 inhibitor, curcumin (and its analogs), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and its analogs; see WO 2008 / 118802), nevitoclax (and its analogs, see US 7,390,799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and its analogs, see WO 2004 / 106328), S-001 (Gloria Pharmaceuticals), the TW series of compounds (Univ. of Michigan), and venetoclax. In some embodiments, the Bcl-2 inhibitor is a small molecule therapeutic. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic.

[1259] As used herein, the term "BTK inhibitor" includes, but is not limited to, compounds that have inhibitory activity against Bruton's tyrosine kinase (BTK), including, but not limited to, AVL-292 and ibrutinib.

[1260] As used herein, the term "SYK inhibitor" includes, but is not limited to, compounds that have inhibitory activity on spleen tyrosine kinase (SYK), including, but not limited to, PRT-062070, R-343, R-333, Excellair, PRT-062607, and fostamatinib.

[1261] Further examples of BTK inhibiting compounds, and further examples of conditions that can be treated by such compounds in combination with the compounds of the invention, can be found in WO2008 / 039218 and WO2011 / 090760, the entire contents of which are incorporated herein by reference.

[1262] Further examples of SYK inhibiting compounds, and further examples of conditions that may be treated by such compounds in combination with the compounds of the invention, can be found in WO 2003 / 063794, WO 2005 / 007623 and WO 2006 / 078846, the entire contents of which are incorporated herein by reference.

[1263] Further examples of PI3K inhibitory compounds, and further examples of conditions that can be treated by such compounds in combination with the compounds of the invention, can be found in WO 2004 / 019973, WO 2004 / 089925, WO 2007 / 016176, US 8,138,347, WO 2002 / 088112, WO 2007 / 084786, WO 2007 / 129161, WO 2006 / 122806, WO 2005 / 113554, and WO 2007 / 044729, the entire contents of which are incorporated herein by reference.

[1264] Further examples of JAK inhibitory compounds, and further examples of conditions that can be treated by such compounds in combination with the compounds of the invention, can be found in WO 2009 / 114512, WO 2008 / 109943, WO 2007 / 053452, WO 2000 / 142246 and WO 2007 / 070514, the entire contents of which are incorporated herein by reference.

[1265] Other anti-angiogenic compounds include compounds that have other mechanisms for their activity, for example, not related to protein or lipid kinase inhibition, such as thalidomide (Thalomidμ) and TNP-470.

[1266] Examples of proteasome inhibitors that can be used in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salisosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[1267] Compounds that target, decrease or inhibit the activity of a protein or lipid phosphatase are, for example, inhibitors of phosphatase 1, phosphatase 2A, or CDC25, such as okadaic acid or a derivative thereof.

[1268] Compounds that induce cell differentiation processes include, but are not limited to, retinoic acid, α-γ- or δ-tocopherol, or α-γ- or δ-tocotrienol.

[1269] The term cyclooxygenase inhibitors as used herein includes, but is not limited to, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and its derivatives, such as celecoxib (Celebrex TM ), rofecoxib (Vioxx TM ), etoricoxib, valdecoxib, or 5-alkyl-2-arylaminophenylacetic acid, for example, 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid and lumiracoxib.

[1270] As used herein, the term "bisphosphonate" includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic, and zoledronic acid. TM Clodronic acid is sold under the trade name Bonefos TM Tiludronic acid is sold under the trade name Skelid TM Pamidronate is sold under the trade name Aredia TM Alendronate is sold under the trade name Fosamax TM Ibandronic acid is sold under the trade name Bondranat TM Actonel TM Zoledronic acid is sold under the trade name ZometaTM The term "mTOR inhibitor" relates to compounds that inhibit the mammalian target of rapamycin (mTOR) and have antiproliferative activity, such as sirolimus ), everolimus (Certican TM ), CCI-779 and ABT578.

[1271] The term "heparanase inhibitor" as used herein refers to a compound that targets, reduces or inhibits the degradation of heparan sulfate. The term includes, but is not limited to, PI-88. The term "biological response modifier" as used herein refers to a lymphokine or interferon.

[1272] As used herein, the term "inhibitor of an oncogenic isoform of Ras," such as H-Ras, K-Ras, or N-Ras, refers to a compound that targets, reduces, or inhibits the oncogenic activity of Ras. For example, a "farnesyl transferase inhibitor," such as L-744832, DK8G557, or R115777 (Zarnestra TM As used herein, the term "telomerase inhibitor" refers to a compound that targets, reduces, or inhibits telomerase activity. Compounds that target, reduce, or inhibit telomerase activity are specifically compounds that inhibit the telomerase receptor, such as telomestatins.

[1273] The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets, reduces or inhibits the activity of methionine aminopeptidase. Compounds that target, reduce or inhibit the activity of methionine aminopeptidase include, but are not limited to, bengamide or its derivatives.

[1274] The term "proteasome inhibitor" as used herein refers to a compound that targets, decreases or inhibits the activity of the proteasome. Compounds that target, decrease or inhibit the activity of the proteasome include, but are not limited to, bortezomib (Velcade TM ) and MLN341.

[1275] As used herein, the term "matrix metalloproteinase inhibitors" or ("MMP" inhibitors) includes, but is not limited to, collagen peptide mimetic and non-peptide mimetic inhibitors, tetracycline derivatives, for example, the hydroxamate peptide mimetic inhibitor batimastat, and its orally bioavailable analogs marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551) BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

[1276] As used herein, the term "compounds for treating hematological malignancies" includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, decrease or inhibit the activity of the FMS-like tyrosine kinase receptor (Flt-3R); interferons, 1-β-D-arabinofuranosylcytosine (ara-c) and busulfan; and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

[1277] Compounds that target, decrease or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R) are specifically compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518.

[1278] As used herein, the term "HSP90 inhibitor" includes, but is not limited to, compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90; compounds that degrade, target, reduce, or inhibit HSP90 client proteins through the ubiquitin proteosome pathway. Compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90, specifically compounds, proteins, or antibodies that inhibit the ATPase activity of HSP90, include, for example, 17-allylamino, 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other geldanamycin-related compounds; radicicol, and HDAC inhibitors.

[1279] As used herein, the term "anti-proliferative antibodies" includes but is not limited to trastuzumab (Herceptin TM ), trastuzumab-DM1, cetuximab (erbitux), bevacizumab (Avastin TM ), rituximab PRO64553 (anti-CD40) and 2C4 antibodies. Antibodies refer to intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[1280] For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia therapies, in particular, in combination with the therapy for the treatment of AML. Specifically, the compounds of the present invention can be co-administered with, for example, a farnesyl transferase inhibitor and / or other drugs that can be used to treat AML, such as, daunorubicin, doxorubicin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatin, and PKC412.

[1281] Other anti-leukemia compounds include, for example, Ara-C, a pyrimidine analog, which is a 2'-α-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine analogs of hypoxanthine, 6-mercaptopurine (6-MP), and fludarabine phosphate. Compounds that target, reduce, or inhibit the activity of histone deacetylase (HDAC) inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), inhibit the activity of enzymes called histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly known as FR901228), trichostatin A and compounds disclosed in US Pat. No. 6,552,065, including but not limited to N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-acrylamide, or a pharmaceutically acceptable salt thereof and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-acrylamide, or a pharmaceutically acceptable salt thereof, particularly lactate. As used herein, a somatostatin receptor antagonist refers to a compound that targets, treats or inhibits somatostatin receptors, such as octreotide and SOM230. Tumor cell destruction methods refer to methods such as ionizing radiation. The term "ionizing radiation" as used herein and hereinbelow refers to ionizing radiation that occurs as electromagnetic rays (e.g., X-rays and gamma rays) or particles (e.g., alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiotherapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., eds., 4th ed., Vol. 1, pp. 248-275 (1993).

[1282] Also included are EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressants that regulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs, including but not limited to fludarabine and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with anti-ALL ara-C) and / or pentostatin. Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[1283] Also included are compounds, proteins or monoclonal antibodies, particularly those directed against VEGF, such as: 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine, or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; Angiostatin TM ;Endostatin TM ; anthranilamide; ZD4190; Zd6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, for example, rhuMAb and RHUFab, or VEGF aptamers, for example Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, Angiozyme (RPI 4610) and bevacizumab (Avastin TM ).

[1284] As used herein, photodynamic therapy refers to therapy that uses certain chemicals known as photosensitizing compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as Visudyne TM and sodium porphyrin.

[1285] As used herein, angiostatic steroids refer to compounds that block or inhibit angiogenesis, such as, for example, anecortave, triamcinolone acetonide, hydrocortisone, 11-α-epihydrocortisol, 11-deoxycortisol, 17α-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone, and dexamethasone.

[1286] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[1287] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormone compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or miscellaneous compounds, or compounds with other or unknown mechanisms of action.

[1288] The structures of active compounds identified by number, generic or trade name may be taken from actual editions of the standard compendium "The Merck Index" or from databases such as Patents International (eg IMS World Publications).

[1289] Exemplary Immuno-Oncology Agents

[1290] In some embodiments, one or more other therapeutic agents are immuno-oncology agents. As used herein, the term "immuno-oncology agent" refers to an agent that effectively enhances, stimulates, and / or upregulates an immune response in a subject. In some embodiments, the administration of an immuno-oncology agent together with a compound of the invention has a synergistic effect in treating cancer.

[1291] The immuno-oncology agent can be, for example, a small molecule drug, an antibody, or a biologic or small molecule. Examples of biologic immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is humanized or human.

[1292] In some embodiments, the immuno-oncology agent is (i) an agonist of a stimulatory (including co-stimulatory) receptor, or (ii) an antagonist of an inhibitory (including co-inhibitory) signal on T cells, both of which result in amplification of antigen-specific T cell responses.

[1293] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, including B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the TNF family of molecules that bind to members of the homologous TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL , TWEAKR / Fn14, TWEAK, BAFFR, EDR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, and NGFR.

[1294] In some embodiments, the immuno-oncology agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines), or a cytokine that stimulates T cell activation for stimulating an immune response.

[1295] In some embodiments, the compound of the present invention and the combination of immune-tumor agent can stimulate T cell response.In some embodiments, immune-tumor agent is:(i) antagonist (for example, immune checkpoint inhibitor) of the protein that suppresses T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4;Or (ii) agonist of protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

[1296] In some embodiments, the immuno-oncology agent is an antagonist of an inhibitory receptor on NK cells, or an agonist of an activating receptor on NK cells. In some embodiments, the immuno-oncology agent is an antagonist of KIR, such as lirilumab.

[1297] In some embodiments, the immuno-oncology agent is an agent that inhibits or depletes macrophages or monocytes, including but not limited to a CSF-1R antagonist, such as a CSF-1R antagonist antibody, including RG7155 (WO2011 / 70024, WO2011 / 107553, WO2011 / 131407, WO2013 / 87699, WO2013 / 119716, WO2013 / 132044) or FPA-008 (WO2011 / 140249; WO2013 / 169264; WO2014 / 036357).

[1298] In some embodiments, the immuno-oncology agent is selected from the group consisting of agonists that bind to positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction)), agents that deplete or suppress Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab, or by in vitro anti-CD25 bead depletion), agents that inhibit metabolic enzymes (e.g., IDO), or reverse / prevent T cell energy or exhaustion), and agents that trigger innate immune activation and / or inflammation at the tumor site.

[1299] In some embodiments, the immuno-oncology agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, the antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab.

[1300] In some embodiments, the immuno-oncology agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immuno-oncology agent is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). In some embodiments, the immuno-oncology agent can be pidilizumab (CT-011). In some embodiments, the immuno-oncology agent is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, referred to as AMP-224.

[1301] In some embodiments, the immuno-oncology agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonist PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[1302] In some embodiments, the immuno-oncology agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonist LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016 (WO2010 / 19570, WO2014 / 08218) or IMP-731 or IMP-321 (WO2008 / 132601, WO2009 / 44273).

[1303] In some embodiments, the immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonist CD137 antibody. In some embodiments, the CD137 antibody is urelumab or PF-05082566 (WO2012 / 32433).

[1304] In some embodiments, the immuno-oncology agent is a GITR agonist. In some embodiments, the GITR agonist is an agonistic GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO2006 / 105021, WO2009 / 009116), or MK-4166 (WO2011 / 028683).

[1305] In some embodiments, the immuno-oncology agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from: epacadostat (INCB024360, Incyte); or indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); an enzyme that breaks down kynurenine (Kynase, Kyn Therapeutics); and NLG-919 (WO2009 / 73620, WO2009 / 1156652, WO2011 / 56652, WO2012 / 142237).

[1306] In some embodiments, the immuno-oncology agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[1307] In some embodiments, the immuno-oncology agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO2006 / 029879).

[1308] In some embodiments, the immuno-oncology agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the immuno-oncology agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[1309] In some embodiments, the immuno-oncology agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is varlilumab.

[1310] In some embodiments, the immuno-oncology agent is MGA271 (directed against B7H3) (WO2011 / 109400).

[1311] In some embodiments, the immuno-oncology agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolizumab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isaxilimab, tuximab), lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[1312] In some embodiments, the immuno-oncology agent is an immunostimulatory agent. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can release activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in a growing number of tumor histologies, including some tumor types that are not generally considered to be sensitive to immunotherapy. See, for example, Okazaki, T et al. (2013) Nat. Immunol. 14, 1212–1218; Zou et al. (2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab ( Bristol-Myers Squibb (formerly known as ONO-4538, MDX1106, and BMS-936558) has shown potential in improving overall survival in patients with renal clear cell carcinoma (RCC) who have experienced disease progression during or after prior anti-angiogenic therapy.

[1313] In some embodiments, the immunomodulatory therapeutic agent specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutic agents that can be used in the present invention include: pomalidomide ( Celgene; lenalidomide ( Celgene; Ingenol Mebutate ( LEO Pharma).

[1314] In some embodiments, the immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from the group consisting of: sipuleucel-T ( Dendreon / Valeant Pharmaceuticals), which has been approved for the treatment of asymptomatic, or minimally symptomatic, metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec ( BioVex / Amgen, formerly known as T-VEC), a genetically modified oncolytic virus therapy approved for the treatment of unresectable skin, subcutaneous, and nodular lesions in melanoma. In some embodiments, the immuno-oncology agent is selected from oncolytic virus therapies, such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly known as Jennerex Biotherapeutics), a thymidine kinase-(TK-) deficient vaccinia virus engineered to express GM-CSF for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep ( Oncolytics Biotech, a variant of a respiratory orphan virus (reovirus), is unable to replicate in cells without RAS activation in a variety of cancers, including colorectal cancer (NCT01622543); prostate cancer (NCT01619813); head and neck squamous cell carcinoma (NCT01166542); pancreatic adenocarcinoma (NCT00998322); and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly ColoAd1), an adenovirus engineered to express full-length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117); metastatic or advanced epithelial tumors, such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly Oncos), an engineered Adenoviruses engineered to express GM-CSF were studied in melanoma (NCT03003676); and peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), vaccinia virus engineered to express β-galactosidase (β-gal) / β-glucuronidase or β-gal / human sodium iodide cotransporter (hNIS), respectively, were studied in peritoneal cancer (NCT01443260), fallopian tube cancer, and ovarian cancer (NCT02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, was studied in bladder cancer (NCT02365818).

[1315] In some embodiments, the immuno-oncology agent is selected from: JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is capable of converting the prodrug 5-fluorocytosine into the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapeutics that target difficult-to-treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus named: Ad5 / 3-E2F-δ24-hTNFα-IRES-hIL20; and VSV-GP (Vira Therapeutics), a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express a protein designed to enhance antigen-specific CD8 + Antigens to which T cells respond.

[1316] In some embodiments, the immuno-oncology agent is a T cell engineered to express a chimeric antigen receptor or CAR. T cells engineered to express this chimeric antigen receptor are called CAR-T cells.

[1317] A CAR consisting of a binding domain has been constructed that can be derived from the following: a natural ligand, a single-chain variable fragment (scFv) of a monoclonal antibody specific for a cell surface antigen, fused to an intracellular domain (endodomain) that is the functional end of a T cell receptor (TCR), such as the CD3-ζ signaling domain from TCR, which is capable of generating activation signals in T lymphocytes. Upon antigen binding, such CARs connect to the endogenous signaling pathway in effector cells and generate activation signals similar to those triggered by the TCR complex.

[1318] For example, in some embodiments, the CAR-T cell is one of those described in U.S. Pat. No. 8,906,682 (incorporated herein by reference in its entirety), which discloses that CAR-T cells are engineered to include an extracellular domain with an antigen binding domain (e.g., a domain that binds to CD19) fused to the intracellular signaling domain of the ζ chain of the T cell antigen receptor complex (e.g., CD3ζ). When expressed in T cells, CARs are able to redirect antigen recognition based on antigen binding specificity. For CD19, the antigen is expressed on malignant B cells. Currently, more than 200 clinical trials using CAR-T are underway in various indications [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[1319] In some embodiments, the immunostimulatory agent is an activator of retinoic acid receptor-related orphan receptor gamma (RORγt). RORγt is a transcription factor that plays a key role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as the differentiation of IL-17-expressing innate immune cell subsets such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT02929862).

[1320] In some embodiments, the immunostimulant is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLR include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG that is being studied for use in B cells, follicular lymphoma, and other lymphomas (NCT02254772). Agonists or activators of TLR8 that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals), which are being studied for use in head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[1321] Other immuno-oncology agents that can be used in the present invention include: urerulumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirelumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences) Sciences), an anti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[1322] In some embodiments, the immunostimulatory agent is selected from evolozumab, mifamortide, an agonist or activator of a toll-like receptor, and an activator of RORγt.

[1323] In some embodiments, the immunostimulatory therapeutic is recombinant human interleukin 15 (rhIL-15). rhIL-15 has been clinically tested as a therapy for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemia (NCT02689453). In some embodiments, the immunostimulatory agent is recombinant human interleukin 12 (rhIL-12). In some embodiments, the IL-15-based immunotherapeutic is heterodimeric IL-15 (hetIL-15, Novartis / Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed with a soluble IL-15 binding protein, the IL-15 receptor alpha chain (IL15:sIL-15RA), which has been tested in Phase 1 clinical trials for melanoma, renal cell carcinoma, non-small cell lung cancer, and head and neck squamous cell carcinoma (NCT02452268). In some embodiments, the recombinant human interleukin 12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724, or NCT02542124.

[1324] In some embodiments, the immuno-oncology agent is selected from those described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pp. 603-622, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is selected from the examples described in Table 1B by Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule that targets an immuno-oncology target selected from those listed in Table 2 by Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule selected from those listed in Table 2 by Jerry L. Adams et al.

[1325] In some embodiments, the immuno-oncology agent is selected from the small molecule immuno-oncology agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, volume 28, pages 319-329, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is an agent that targets a pathway as described by Peter L. Toogood.

[1326] In some embodiments, the immuno-oncology agent is selected from Sandra L. Ross et al., "Bispecific T cell engager Antibody constructs can mediate bystander tumor cell killing", PLoS ONE 12(8):e0183390, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is a bispecific T cell engager (engager) Antibody Constructs. In some embodiments, the bispecific T cell engager The antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager The antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager Antibody constructs activate T cells. In some embodiments, bispecific T cell engagers The antibody construct activates T cells, which release cytokines that induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager The antibody construct activates T cells, which results in induced bystander cell lysis. In some embodiments, the bystander cells are in a solid tumor. In some embodiments, the lysed bystander cells are adjacent to the -activated T cells. In some embodiments, the adjacent cells comprise tumor-associated antigen (TAA) negative cancer cells. In some embodiments, the adjacent cells include EGFR-negative cancer cells. In some embodiments, the immune-oncology agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immune-oncology agent is a tumor-infiltrating T cell that is expanded ex vivo. In some embodiments, the immune-oncology agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly connects T cells to tumor-associated surface antigens (TAA).

[1327] Exemplary immune checkpoint inhibitors

[1328] In some embodiments, the immuno-oncology agent is an immune checkpoint inhibitor described herein.

[1329] As used herein, the term "checkpoint inhibitor" refers to an agent that can be used to prevent cancer cells from escaping the patient's immune system. One of the main mechanisms of anti-tumor immune subversion is called "T-cell exhaustion", which is caused by upregulation of inhibitory receptors due to chronic exposure to antigens. These inhibitory receptors serve as immune checkpoints to prevent uncontrolled immune responses.

[1330] PD-1 and co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin and mucin domain-containing-3 (Tim-3), and lymphocyte activation gene-3 (Lag-3; CD223), are commonly referred to as checkpoint regulators. They act as molecular "gatekeepers" to allow extracellular information to determine whether cell cycle progression and other intracellular signaling processes should proceed.

[1331] In some embodiments, the immune checkpoint inhibitor is an antibody directed against PD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) to prevent the receptor from binding to its inhibitory ligand, PDL-1, thereby overriding the tumor's ability to suppress the host's anti-tumor immune response.

[1332] In one aspect, the checkpoint inhibitor is a biotherapeutic agent or a small molecule. In another aspect, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. In another aspect, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In another aspect, the check point inhibitor interacts with a ligand selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or a combination thereof. In one aspect, the check point inhibitor is an immunostimulant, a T cell growth factor, an interleukin, an antibody, a vaccine, or a combination thereof. In another aspect, the interleukin is IL-7 or IL-15. In a specific aspect, the interleukin is a glycosylated IL-7. In another aspect, the vaccine is a dendritic cell (DC) vaccine.

[1333] Checkpoint inhibitors include any agent that blocks or inhibits immune system inhibitory pathways in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies that bind to and block or inhibit immune checkpoint receptors, or antigen binding fragments thereof, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is present in all NK, γδ and memory CD8 +(αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[1334] In certain embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from nivolumab. Ipilimumab and pembrolizumab In some embodiments, the checkpoint inhibitor is selected from: nivolumab (anti-PD-1 antibody, Bristol-Myers Squibb; pembrolizumab (anti-PD-1 antibody, Merck); Ipilimumab (anti-CTLA-4 antibody, Bristol-Myers Squibb; durvalumab (anti-PD-L1 antibody, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, Genentech).

[1335] In some embodiments, the checkpoint inhibitor is selected from the group consisting of: lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirlumab, IPH2101, pembrolizumab and tremelimumab.

[1336] In some embodiments, the immune checkpoint inhibitor is: REGN2810 (Regeneron), an anti-PD-1 antibody that has been tested in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1 in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avastin ( Pfizer / Merck KGaA), also known as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody in clinical trials for: non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer; or PDR001 (Novartis), an inhibitory antibody that binds to PD-1 in clinical trials for: non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; AstraZeneca) is a fully human monoclonal antibody against CTLA-4 that has been studied in clinical trials for multiple indications, including mesothelioma, colorectal cancer, renal cancer, breast cancer, lung cancer, non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, liver metastases, hepatocellular carcinoma, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody that is being studied in a Phase 1 clinical trial for advanced solid tumors (NCT02694822).

[1337] In some embodiments, the checkpoint inhibitor is an inhibitor of T-cell immunoglobulin mucin (TIM-3) containing protein-3. TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody that is being studied in advanced malignancies (NCT02608268).

[1338] In some embodiments, the checkpoint inhibitor is an inhibitor of a T cell immunoreceptor with Ig and ITIM domains, or TIGIT (an immunoreceptor on certain T cells and NK cells). TIGIT inhibitors that can be used in the present invention include BMS-986207 (Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428).

[1339] In some embodiments, the checkpoint inhibitor is an inhibitor of Lymphocyte Activation Gene-3 (LAG-3). LAG-3 inhibitors that can be used in the present invention include BMS-986016, REGN3767, and IMP321. BMS-986016 (Bristol-Myers Squibb) is an anti-LAG-3 antibody that is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody that is being studied in malignant tumors (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein that is being studied in the following: melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[1340] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists being studied in clinical trials include: PF-04518600 / PF-8600 (Pfizer), an agonist anti-OX40 antibody for metastatic renal cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonist anti-OX40 antibody in a Phase 1 cancer trial (NCT02528357); MEDI0562 (Medimmune / AstraZeneca) ca), an agonist anti-OX40 antibody, in patients with advanced solid tumors (NCT02318394 and NCT02705482); MEDI6469, an agonist anti-OX40 antibody (Medimmune / AstraZeneca), in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb), an agonist anti-OX40 antibody, in patients with advanced cancers (NCT02737475).

[1341] Checkpoint inhibitors that can be used in the present invention include CD137 (also known as 4-1BB) agonists. CD137 agonists being studied in clinical trials include: utomilumab (PF-05082566, Pfizer), an agonistic anti-CD137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and neoplasms (NCT02554812 and NCT05082566); Urecitabine (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD137 antibody, for melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981).

[1342] Checkpoint inhibitors that can be used in the present invention include CD27 agonists. CD27 agonists being studied in clinical trials include: Valirumab (CDX-1127, Celldex Therapeutics), an agonist anti-CD27 antibody, for head and neck squamous cell carcinoma, ovarian cancer, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[1343] Checkpoint inhibitors useful in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists being studied in clinical trials include TRX518 (Leap Therapeutics), an agonist anti-GITR antibody for malignant melanoma and other solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonist anti-GITR antibody for solid tumors and lymphomas (NCT02740270); INCAGN01876 (Incyte / Agenus), an agonist anti-GITR antibody for advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonist anti-GITR antibody for solid tumors (NCT02132754); and MEDI1873 (Medimmune / AstraZeneca), a human IgG1 An agonistic hexameric GITR-ligand molecule with an Fc domain for use in advanced solid tumors (NCT02583165).

[1344] Checkpoint inhibitors that can be used in the present invention include inducible T-cell co-stimulator (ICOS, also known as CD278) agonists. ICOS agonists being studied in clinical trials include: MEDI-570 (Medimmune), an agonistic anti-ICOS antibody, used in lymphoma (NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody, used in Phase 1 (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody, used in Phase 1 (NCT02904226).

[1345] Checkpoint inhibitors useful in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include lirelumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody, in leukemias (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma), in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to three domains of the long cytoplasmic tail (KIR3DL2), in lymphoma (NCT02593045).

[1346] Checkpoint inhibitors useful in the present invention include CD47 inhibitors of the interaction between CD47 and signal regulatory protein alpha (SIRPα). CD47 / SIRPα inhibitors being studied in clinical trials include: ALX-148 (Alexo Therapeutics), an antagonist variant of SIRPα that binds to CD47 and blocks CD47 / SIRPα-mediated signaling, in Phase 1 (NCT03013218); TTI-621 (SIRPα-Fc, Trillium Therapeutics), a soluble recombinant fusion protein generated by linking the N-terminal CD47-binding domain of SIRPα to the Fc domain of human IgG1 that works by binding to human CD47 and blocking its delivery of a “don’t eat” signal to macrophages, in Phase 1 (NCT02890368 and NCT02663518); CC-90002 (Celgene), an anti-CD47 antibody, in leukemia (NCT02641002); and Hu5F9-G4 (Forty-three Therapeutics). Seven, Inc.), in colorectal and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[1347] Checkpoint inhibitors that can be used in the present invention include CD73 inhibitors. CD73 inhibitors being studied in clinical trials include: MEDI9447 (Medimmune), an anti-CD73 antibody, in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody, in solid tumors (NCT02754141).

[1348] Checkpoint inhibitors useful in the present invention include agonists of the stimulator of interferon genes protein (STING, also known as transmembrane protein 173 or TMEM173). STING agonists being studied in clinical trials include: MK-1454 (Merck), an agonist synthetic cyclic dinucleotide, in lymphoma (NCT03010176); and ADU-S100 (MIW815, AduroBiotech / Novartis), an agonist synthetic cyclic dinucleotide, in Phase 1 (NCT02675439 and NCT03172936).

[1349] Checkpoint inhibitors that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors being studied in clinical trials include: pexidartinib (PLX3397, Plexxikon), a small molecule inhibitor of CSF1R for colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710) and melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, gastrointestinal stromal tumors (GIST) and ovarian cancer (NCT02452424); and IMC-CS4 (LY3022855 , Lilly), an anti-CSF-1R antibody for pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid carboxamide, Novartis), an orally available CSF1R inhibitor for advanced solid tumors (NCT02829723).

[1350] Checkpoint inhibitors that can be used in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors being studied in clinical trials include monazumab (IPH2201, Innate Pharma), an anti-NKG2A antibody for head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[1351] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, afocizumab, durvalumab, atuzumab, or pidilizumab.

[1352] Therapeutic uses

[1353] The bicyclic peptide of the present invention has a specific use as a nectin-4 binding agent.

[1354] Integrin-4 is a surface molecule that belongs to the integrin protein family, which includes four members. Integrins are cell adhesion molecules that play key roles in various biological processes in epithelial, endothelial, immune, and neuronal cells during development and adulthood, such as polarity, proliferation, differentiation, and migration. They are involved in several human pathologies. They serve as the primary receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding integrin-1 (PVRL1) or integrin-4 (PVRL4) lead to ectodermal dysplasia syndromes associated with other abnormalities. Integrin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Integrin-4 is a tumor-associated antigen, accounting for 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively, primarily in tumors with poor prognosis. Its expression is not detected in corresponding normal tissues. In breast tumors, integrin-4 is predominantly expressed in triple-negative and ERBB2+ cancers. In the serum of patients with these cancers, soluble forms of integrins-4 have been detected and are associated with poor prognosis. Serum integrins-4 levels increase during metastasis and decrease after treatment. These results suggest that integrins-4 may be a reliable target for treating cancer. Therefore, several anti-integrins-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting integrins-4 that is currently being studied clinically for the treatment of patients with solid tumors.

[1355] Polypeptide ligands selected according to the methods of the present invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, and the like. Ligands with selected levels of specificity can be used in tests involving non-human animals where cross-reactivity is desired, or in diagnostic applications where cross-reactivity with homologs or paralogs needs to be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response to a predetermined range of antigens can be exploited to tailor vaccines against specific diseases and pathogens.

[1356] For mammalian administration, substantially pure peptide ligands having at least 90 to 95% homogeneity are preferred, and for pharmaceutical administration (particularly when the mammal is a human), 98 to 99% or greater homogeneity is most preferred. After purification, partial purification, or to the desired homogeneity, the selected polypeptide can be used for diagnosis or therapy (including in vitro), or for developing and performing assay procedures, immunofluorescence staining, etc. (Lefkovite and Pernis, (1979 and 1981), Immunological Methods, Volumes I and II, Academic Press, New York).

[1357] According to another aspect of the present invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, inhibiting or treating a disease or condition mediated by Integrin-4.

[1358] According to another aspect of the present invention, there is provided a method of preventing, inhibiting or treating a disease or condition mediated by Integrin-4, the method comprising administering to a patient in need thereof an effector group of a peptide ligand and a drug conjugate as defined herein.

[1359] In one embodiment, the binding protein-4 is mammalian binding protein-4. In another embodiment, the binding protein-4 is human binding protein-4.

[1360] In one embodiment, the disease or condition mediated by Integrin-4 is selected from viral infection, ectodermal dysplasia syndrome and other abnormalities, breast, ovarian and lung cancer, metastatic progression and solid tumors.

[1361] In another embodiment, the disease or condition mediated by Integrin-4 is selected from cancer.

[1362] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous carcinomas, transitional cell carcinomas, and other cancers), such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including esophagus, stomach (stomach), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (e.g., tongue cancer, buccal cancer, larynx cancer, pharynx cancer, nasopharynx cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penile cancer, cervix cancer, carcinoma of the uterus, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin and appendage cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, hyperplastic nevus); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous hematologic disorders, as well as borderline malignancies, including hematologic malignancies and related disorders of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer (NK) NK-cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), as well as hematologic malignancies and related disorders of the myeloid lineage (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome, myelodysplastic disorders, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma sarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas, and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma);and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or congenital or other syndromes that predispose patients to malignancy (e.g., xeroderma).

[1363] In another embodiment, the cancer is selected from hematopoietic malignancies, for example selected from: non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[1364] In yet another embodiment, the cancer is selected from lung cancer (e.g., non-small cell lung cancer), bladder cancer, pancreatic cancer, and breast cancer. The data herein are presented in Examples 1 to 5, which demonstrate that selected bicyclic drug conjugates of the present invention exhibit anti-tumor activity in these cancer models.

[1365] As used herein, the term "prevention" refers to administration of a protective composition prior to the induction of a disease. "Suppression" refers to administration of a composition after the induction event but prior to the clinical appearance of the disease. "Treatment" refers to administration of a protective composition after symptoms of the disease become apparent.

[1366] 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 allow the development of polypeptide ligands that can cross-react with both human and animal targets, thereby allowing the use of animal models.

[1367] In addition, the data provided herein demonstrate an association between copy number variation (CNV) and gene expression of integrin-4 from a variety of tumor types. Thus, according to another aspect of the present invention, there is provided a method for preventing, inhibiting or treating cancer comprising administering to a patient in need thereof an effector group and a drug conjugate of a peptide ligand as defined herein, wherein the patient is identified as having increased copy number variation (CNV) of integrin-4.

[1368] In one embodiment, the cancer is selected from those cancers identified herein as having an increased CNV of Integrin-4. In another embodiment, the cancer is selected from those cancers identified herein as having an increased CNV of Integrin-4, namely: breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate cancer, and gastric cancer.

[1369] The present invention is further described below with reference to the following examples.

[1370] Example

[1371] Abbreviations

[1372] 1,2,4-TriAz 3-(1,2,4-triazol-1-yl)-alanine

[1373] 1Nal 1-naphthylalanine

[1374] 2FuAla 2-furanolalanine

[1375] 2MePhe 2-methyl-phenylalanine

[1376] 2Nal 2-naphthylalanine

[1377] 2Pal 2-pyridylalanine

[1378] 3,3-DPA 3,3-diphenylalanine

[1379] 3MePhe 3-methyl-phenylalanine

[1380] 3Pal 3-pyridylalanine

[1381] 4,4-BPA 4,4-biphenylalanine

[1382] 4,4-DFP 4,4-difluoroproline

[1383] 4MePhe 4-methyl-phenylalanine

[1384] 4Pal 4-pyridylalanine

[1385] 4ThiAz β-(4-thiazolyl)-alanine

[1386] 5FTrp 5-Fluoro-L-tryptophan

[1387] Agb 2-amino-4-guanidinobutyric acid

[1388] Aib aminoisobutyric acid

[1389] AzaTrp Azatryptophan

[1390] Azetidine

[1391] C5A Cyclopentylglycine

[1392] Cha 3-cyclohexyl-alanine

[1393] Cpa cyclopropylalanine

[1394] Cya Cysteic Acid

[1395] DOPA 3,4-dihydroxyphenylalanine

[1396] HArg homoarginine

[1397] HGln High Glutamine

[1398] Hleu homoleucine

[1399] Hphe Homophenylalanine

[1400] Hse(me) Homoserine(Me)

[1401] Hser homoserine

[1402] HyP Hydroxyproline

[1403] Lys(Ac) Lysine (Acetyl)

[1404] Met(O2) methionine sulfone

[1405] Nle norleucine

[1406] Oic octahydroindole carboxylic acid

[1407] Oxa oxazolidine-4-carboxylic acid

[1408] pCoPhe p-carboxy-phenylalanine

[1409] PheOPhe 4-phenoxy-phenylalanine

[1410] Phg phenylglycine

[1411] Pip pipecolic acid

[1412] Pro(4NH) 4-amino-proline

[1413] tBuAla tert-Butyl-alanine

[1414] TetraZ Tetrazolyl Alanine

[1415] Thiamin

[1416] THP(O) Tetrahydropyran-4-propionic acid

[1417] THP(SO2) dioxy-4-tetrahydrothiopyranyl acetic acid

[1418] Trp(Me) methyltryptophan

[1419] Materials and methods

[1420] Peptide synthesis

[1421] Peptide synthesis is based on Fmoc chemistry, using Symphony peptide synthesizers produced by Peptide Instruments and Syro II synthesizers produced by MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) are used, with appropriate side chain protecting groups: in each case, applicable standard coupling conditions are used, followed by deprotection using standard methods.

[1422] Alternatively, peptides were purified using HPLC and modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma) after isolation. To do this, the linear peptide was diluted to ~35 mL with 50:50 MeCN:H2O, ~500 μL of 100 mM TATA in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed at room temperature for approximately 30–60 minutes and lyophilized immediately upon completion (as judged by MALDI). Upon completion, 1 ml of 1 M L-cysteine ​​hydrochloride monohydrate (Sigma) in H2O was added to the reaction for ~60 minutes at room temperature to quench any excess TATA.

[1423] After lyophilization, the modified peptide was purified as above, using a Gemini C18 column (Phenomenex) instead of a Luna C8 column and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA-modified material were pooled, lyophilized, and stored at -20°C.

[1424] Unless otherwise stated, all amino acids were used in the L-configuration.

[1425] In some cases, the peptide was converted to an activated disulfide prior to coupling to a free thiol group of the toxin using the following method: A solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol eq) was added to a solution of the peptide (20 mM) in dry DMSO (1 mol eq). The reaction was mixed thoroughly and DIPEA (20 mol eq) was added. The reaction was monitored by LC / MS until completion.

[1426] Preparation of bicyclic peptide drug conjugates

[1427] Preparation of BCY7826

[1428] Separation conditions: Phase A: 0.075% TFA in H2O, Phase B: MeCN

[1429] Separation method: 18-48-55 minutes, RT = 53.5 minutes

[1430] Separation columns: Luna 200*25mm 10μm, C18, 110A and Gemin 150*30mm, C18, 5μm, 110A, connected, 50℃

[1431] Dissolution method: DMF

[1432] Separation purity: 95%

[1433]

[1434] The peptide was synthesized by solid phase synthesis. 1.11 g of Rink Amide MBHA resin (sub: 0.45 mmol / g) was used. DMF (20 mL) was added to a mixture containing Rink Amide MBHA (0.5 mmol, 1.11 g, 0.45 mmol / g) and Fmoc-Cys(Trt)-OH (0.87 g, 1.5 mmol, 3.0 eq), followed by DIC (3.0 eq) and HOAt (3.0 eq) and mixed for 1 hour. Deblocking was performed using a 20% piperidine solution in DMF. Other amino acids were coupled at 3.0 eq using a DMF (20 mL) solution of the activating agents DIC (3.0 eq) and HOAt (3.0 eq). The reaction was monitored by ninhydrin color reaction or tetrachloroethylene color reaction. After completion of the synthesis, the peptide resin was washed with DMF × 3, MeOH × 3, and then dried overnight under N2 bubbling. Afterwards, the peptide resin was treated with 92.5% TFA / 2.5% TIS / 2.5% EDT / 2.5% H2O for 3 hours. The peptide was precipitated with cold isopropyl ether (200 mL) and centrifuged (3000 rpm for 3 minutes). Isopropyl ether (200 mL) was used to wash the peptide twice. The crude peptide was dried under vacuum for 2 hours. Using the crude peptide (i.e., after peptide cleavage and isopropyl ether precipitation, the precipitate was lyophilized to remove residual isopropyl ether and TFA), the lyophilized powder (0.5 mmol) was dissolved in 500 ml ACN / H2O (50:50), followed by addition of 5 ml of 100 mM TATA. 10 ml of ammonium bicarbonate in H2O (1 M) solution was added and mixed for 1 hour. After the cyclization was complete, the reaction must be quenched with 10.0 eq of cysteine ​​on TATA. At least 5 ml of 1 M cysteine ​​was added to the solution, mixed and allowed to stand for one hour. The solution was lyophilized to obtain a crude product. The crude peptide was purified by preparative HPLC and lyophilized to give the product BCY7814 (144.1 mg, 97.1% purity; 9.6% yield) as a white solid.

[1435] (HPLC analysis conditions)

[1436] Instrument - Agilent 1200HPLC-BE(1-614)

[1437]

[1438]

[1439]

[1440] To a solution of BCY7814 (61.80 mg, 21.29 μmol, 1.1 eq) in DMA (4 mL) was added DIEA (7.50 mg, 58.05 μmol, 10.11 μL, 3.0 eq) and DM1-SO3H-SPDB-NHS. The mixture was stirred at 25°C for 16 hours. LC-MS indicated complete consumption of DM1-SO3H-SPDB-NHS, and a major peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY7826 (0.0242 g, 6.31 μmol, 32.63% yield, 99.7% purity) was obtained as a white solid. Retention time = 13.99 min, indicated mass = 1254.1 (M / 3+1).

[1441] Preparation of BCY8549

[1442] Separation conditions: Phase A: 0.075% TFA in H2O, Phase B: MeCN

[1443] Separation method: 18-48-55 minutes, RT = 53.5 minutes

[1444] Separation column: Luna 200 * 25mm 10μm, C18, 110A and Gemin150 * 30mm, C18, 5μm, 110A, connected, 50℃

[1445] Dissolution method: DMF

[1446] Separation purity: 95%

[1447] BCY8234 was synthesized by solid-phase synthesis.

[1448]

[1449]

[1450] Preparation of compound 2

[1451] The peptide was synthesized using standard Fmoc chemistry.

[1452] 1) DCM was added to a vessel containing CTC resin (5 mmol, 4.3 g, 1.17 mmol / g) and Fmoc-Cit-OH (2.0 g, 5 mmol, 1.0 eq) and bubbled with N2.

[1453] 2) DIEA (4.0 eq) was added dropwise and mixed for 2 hours.

[1454] 3) Add MeOH (5 mL) and mix for 30 minutes.

[1455] 4) Drain and wash with DMF 5 times.

[1456] 5) Add 20% piperidine / DMF and react for 30 minutes.

[1457] 6) Drain and wash with DMF 5 times.

[1458] 7) Add Fmoc-amino acid solution and mix for 30 seconds, then add activation buffer and bubble N2 for about 1 hour.

[1459] 8) Repeat steps 4 to 7 above to couple the following amino acids.

[1460] Notice:

[1461]

[1462] 20% piperidine in DMF was used for Fmoc deprotection for 30 min. The coupling reaction was monitored by ninhydrin test and the resin was washed 5 times with DMF.

[1463] Peptide cleavage and purification:

[1464] 1) Add lysis buffer (20% TFIP / 80% DCM) to the flask containing the side-chain protected peptide at room temperature and stir twice for 1 hour.

[1465] 2) Filter and collect the filtrate.

[1466] 3) Concentrate to remove the solvent.

[1467] 4) The crude peptide was lyophilized to obtain the final product (1.4 g, 85.0% yield).

[1468] Preparation of compound 3

[1469] To a solution of compound 2 (1.65 g, 5.01 mmol, 1.0 eq) in DCM (30 mL) and MeOH (15 mL) was added EEDQ (2.48 g, 10.02 mmol, 2.0 eq) and (4-aminophenyl)methanol (740.37 mg, 6.01 mmol, 1.2 eq). The mixture was stirred at 15 ° C for 16 hours. LC-MS showed that compound 2 was completely consumed and a main peak with the desired m / z was detected. TLC showed that compound 2 was completely consumed and many new spots were formed. The reaction mixture was concentrated under reduced pressure to remove the solvent to obtain a residue. The product was purified by flash silica gel chromatography ( 80 The residue was purified using a Silica Flash Column (gradient elution: 0-15 DCM / MeOH @ 60 mL / min) to obtain compound 3 (1.3 g, 2.99 mmol, 59.72% yield) as a yellow solid.

[1470] Preparation of compound 4

[1471] To a solution of compound 3 (1.3 g, 2.99 mmol, 1.0 eq) in DMF (10 ml) was added DIEA (2.32 g, 17.95 mmol, 3.13 mL, 6.0 eq) and bis(4-nitrophenyl) carbonate (3.64 g, 11.97 mmol, 4.0 eq). The mixture was stirred at 15 ° C for 1 hour. LC-MS showed that compound 3 was completely consumed, and a major peak with the desired m / z was detected. The residue was purified by preparative HPLC (neutral conditions). Compound 4 (1.0 g, 1.67 mmol, 55.74% yield) was obtained as a yellow solid.

[1472] Preparation of compound 5

[1473] To a solution of compound 5 (250.53 mg, 417.84 μmol, 1.5 eq) in DMF (5 mL) were added HOBt (56.46 mg, 417.84 μmol, 1.5 eq) and DIEA (108.01 mg, 835.68 μmol, 145.56 μL, 3.0 eq) and MMAE (0.200 g, 278.56 μmol, 1.0 eq). The mixture was stirred at 35 ° C for 12 hours. LC-MS showed that MMAE was completely consumed and a major peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5 (0.180 g, 152.74 μmol, 54.83% yield) was obtained as a yellow solid.

[1474] Preparation of compound 6

[1475] To a solution of compound 5 (0.170 g, 144.26 μmol, 1.0 eq) in THF (5 mL) and H2O (5 mL) was added LiOH.H2O (12.11 mg, 288.51 μmol, 2.0 eq). The mixture was stirred at 15 ° C for 1 hour. LC-MS showed that compound 5 was completely consumed, and a main peak with the desired m / z was detected. PH=7 was adjusted using AcOH, and THF was removed under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (neutral conditions). Compound 6 (0.185 g, crude product) was obtained as a yellow solid.

[1476] Preparation of BCY8549

[1477] To a solution of compound 6 (0.100 g, 86.93 μmol, 1.0 eq) in DMA (4 mL) were added HOSu (10.00 mg, 86.93 μmol, 1.0 eq) and EDCI (16.66 mg, 86.93 μmol, 1.0 eq). Following NHS ester formation, β-Ala-BCY8234 (525.98 mg, 173.85 μmol, 2.0 eq) and DIEA (33.70 mg, 260.78 μmol, 45.42 μL, 3.0 eq) were added. The mixture was stirred at 15°C for 4 hours. LC-MS showed complete consumption of compound 6, with the detection of a major peak with the desired m / z. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8549 (0.0528 g, 12.15 μmol, 13.98% yield, 95.70% purity) was obtained as a white solid. Retention time = 11.48 minutes. Display mass = 1386.4 (M / 3+H).

[1478] Preparation of BCY8245

[1479] Separation conditions: Phase A: 0.075% TFA in H2O, Phase B: MeCN

[1480] Separation method: 18-48-55 minutes, RT = 53.5 minutes

[1481] Separation columns: Luna 200*25mm 10um, C18, 110A and Gemin 150*30mm, C18, 5um, 110A, connected, 50℃

[1482] Dissolution method: DMF

[1483] Separation purity: 95%

[1484] BCY8234 was synthesized by solid-phase synthesis.

[1485] The reaction diagram of BCY8245 is shown below

[1486]

[1487]

[1488]

[1489] Preparation of compound 3

[1490] Compound 3 was synthesized by a solid phase method.

[1491] Preparation of compound 4

[1492] To a solution of compound 3 (1.3 g, 3.23 mmol, 1.0 eq) in DCM (10 mL) and MeOH (5 mL) was added EEDQ (1.60 g, 6.46 mmol, 2.0 eq) and (4-aminophenyl)methanol (517.16 mg, 4.20 mmol, 1.3 eq). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that compound 3 was completely consumed and a major peak with the desired m / z was detected. The solvent was removed under reduced pressure. The product was purified by flash silica gel chromatography ( 40g The residue was purified by silica flash column, gradient elution (0-15% DCM / MeOH @ 40 mL / min) to obtain compound 4 (0.950 g, 1.87 mmol, 57.94% yield) as a yellow solid.

[1493] Preparation of compound 5

[1494] To a solution of compound 4 (0.950 g, 1.87 mmol, 1.0 eq) in DMF (5 ml) was added DIEA (1.21 g, 9.36 mmol, 1.63 mL, 5.0 eq) and bis(4-nitrophenyl) carbonate (2.28 g, 7.49 mmol, 4.0 eq). The mixture was stirred at 20 ° C for 1 hour. LC-MS showed that compound 4 was completely consumed, and a major peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5 (0.400 g, 594.64 μmol, 31.77% yield) was obtained as a white solid.

[1495] Preparation of compound 6

[1496] To a solution of compound 5 (0.200 g, 297.32 μmol, 1.0 eq) in DMF (5 mL) were added HOBt (52.23 mg, 386.51 μmol, 1.3 eq) and DIEA (115.28 mg, 891.95 μmol, 155.36 μL, 3.0 eq) and MMAE (192.12 mg, 267.59 μmol, 0.9 eq). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that compound 5 was completely consumed, and a major peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 6 (0.160 g, 127.84 μmol, 43.00% yield) was obtained as a white solid.

[1497] Preparation of compound 7

[1498] To a solution of compound 6 (0.160 g, 127.84 μmol, 1.0 eq) in THF (3 mL) and H₂O (3 mL) was added LiOH.H₂O (26.82 mg, 639.21 μmol, 5.0 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed complete consumption of compound 6, with a major peak with the desired m / z detected. THF was removed under reduced pressure, and the pH was adjusted to 7 with AcOH, and the mixture was lyophilized. Compound 7 (0.130 g, 105.05 μmol, 82.17% yield) was obtained as a white solid.

[1499] Preparation of compound 8

[1500] To a solution of compound 7 (36.27 mg, 315.15 μmol, 3.0 eq) in DMA (6 mL) and DCM (2 mL) was added EDCI (60.41 mg, 315.15 μmol, 3.0 eq). The mixture was stirred at 15 ° C for 3 hours. LC-MS showed that compound 7 was completely consumed, and a main peak with the desired m / z was detected. DCM was removed under reduced pressure. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 8 (0.095 g, 71.18 μmol, 67.76% yield) was obtained as a white solid.

[1501] Preparation of BCY8245

[1502] To a solution of BCY8234 (66.41 mg, 22.48 μmol, 1.0 eq) in DMA (4 mL) was added DIEA (8.72 mg, 67.44 μmol, 11.75 μL, 3.0 eq) and compound 8 (0.030 g, 22.48 μmol, 1.0 eq). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that BCY8234 was completely consumed, and a major peak with the desired m / z or desired mass was detected. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8245 (0.0427 g, 10.16 μmol, 45.19% yield, 99.30% purity) was obtained as a white solid. Retention time = 11.7 minutes. Display mass (Mass found) = 1043.9 (M / 4+H).

[1503] The following bicyclic drug conjugates were prepared in a similar manner to BCY8245:

[1504] Table 1A

[1505]

[1506] Biological data

[1507] Integrin-4 direct binding assay

[1508] The affinity (Ki) of the peptides of the present invention for human nectin-4 was determined using fluorescence polarization assay according to the method disclosed in WO 2016 / 067035. The peptides of the present invention with fluorescent tags (fluorescein, SIGMA or Alexa Fluor 488 TM, Fisher Scientific) was diluted to 2.5 nM in PBS (containing 0.01% Tween 20) or in 50 mM HEPES (containing 100 mM NaCl and 0.01% Tween pH 7.4) (both referred to as assay buffer). This was combined with the titrated protein in the same assay buffer as the peptide to give a total volume of 25 μL of 1 nM peptide in a black-walled, low-volume, low-binding 384-well plate, typically 5 μL of assay buffer, 10 μL of protein, and then 10 μL of fluorescent peptide. A 1:2 serial dilution was used to give 12 different concentrations, ranging from 500 nM for known high-affinity binders to 10 μM for low-affinity binders and selectivity assays. Measurements were performed on a BMG PHERAstar FS equipped with an "FP 485 520 520" optical module, which excites at 485 nm and detects parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25°C with 200 flashes per well and a 0.1 second localization delay, and each well was measured for 60 minutes at intervals of 5 to 10 minutes. At the end of 60 minutes, in the absence of protein in the well, the gain for the analysis was determined for each tracer. Data were analyzed using Systat Sigmaplot version 12.0. mP values ​​were fitted to a user-defined quadratic equation to generate Kd values: f = ymin + (ymax - ymin) / Lig * ((x + Lig + Kd) / 2 - sqrt ((((x + Lig + Kd) / 2)^2) - (Lig * x))). "Lig" is the defined value for the tracer concentration used.

[1509] Integrin-4 competition binding assay

[1510] The peptides without fluorescent tags were tested for competition with ACPFGCHTDWSWPIWCA-Sar6-K(F1) (SEQ ID NO: 217) (Kd = 5 nM - determined using the protocol described above). Peptides were diluted to the appropriate concentration in assay buffer using a maximum of 5% DMSO followed by a 1 to 2 serial dilution as described for the direct binding assay. 5 μL of the diluted peptide was added to the plate, followed by 10 μL of human nectin-4 and then 10 μL of the fluorescent peptide. Measurements were performed as in the direct binding assay, however, the gain was determined prior to the first measurement. Data were analyzed using Systat Sigmaplot version 12.0, where mP values ​​were fitted to a user-defined cubic equation to generate Ki values:

[1511] f=ymin+(ymax-ymin) / Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Pro t*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(K comp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Com p-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-P rot*c))) / ((3*Klig)+((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig* Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig- Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Comp-Prot*c)^ 2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-Prot*c)))).

[1512] "Lig", "KLig" and "Prot" are defined values ​​related to the concentration of fluorescent peptide, Kd of fluorescent peptide and bindin concentration, respectively.

[1513] Integrin-4 Biacore SPR binding assay

[1514] Biacore experiments were performed to determine the k of monomeric peptides binding to human Necin-4 protein. a (M-1 s -1 ), k d (s -1 ), K D (nM) values ​​(obtained from Charles River).

[1515] Human integrin-4 (residues Gly32-Ser349; NCBI reference sequence: NP_112178.2) with a gp67 signal sequence and a C-terminal FLAG tag was cloned using standard Bac-to-Bac TM The protocol (Life Technologies) was cloned into pFastbac-1 and baculovirus. P1 virus stock was used to infect 1×10 cells in Excell-420 medium (Sigma) at 27°C with an M01 of 2. 6 ml -1 Sf21 cells were cultured and the supernatant was harvested at 72 hours. The supernatant was bound in batches to anti-FLAG M2 affinity agarose resin (Sigma) washed in PBS for 1 hour at 4°C, and the resin was then transferred to a column and washed thoroughly with PBS. The protein was eluted with 100 pg / ml of FLAG peptide. The eluted protein was concentrated to 2 ml and loaded onto an S-200 Superdex column (GE Healthcare) in PBS at a speed of 1 ml / min. 2 ml fractions were collected and the fraction containing the integrin-4 protein was concentrated to 16 mg / ml.

[1516] Follow the manufacturer's recommended operating procedures to use EZ-Link TM Proteins were randomly biotinylated in PBS using Sulfo-NHS-LC-LC-biotin reagent (Thermo Fisher). Proteins were extensively desalted using spin columns to remove uncoupled biotin into PBS.

[1517] In order to analyze peptide binding, a Biacore 3000 instrument was used, which utilizes a CM5 chip (GE Healthcare). Using standard amine coupling chemistry, at 25 ° C, with HBS-N (10mM HEPES, 0.15M NaCl, pH 7.4) as a running buffer, streptavidin was fixed on the chip. In brief, 0.4M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1M N-hydroxysuccinimide (NHS) in a 1: 1 ratio was injected at a flow rate of 10 μl / min for 7 minutes to activate the carboxymethyl dextran surface. In order to capture streptavidin, the protein was diluted to 0.2 mg / ml in 10mM sodium acetate (pH 4.5) and captured by injecting 120 μl of streptavidin onto the activated chip surface. Residual activated groups were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 minutes, and biotinylated bindin-4 was captured to a level of 1,200-1,800 RU. The buffer was changed to PBS / 0.05% Tween 20, and a series of peptide dilutions were prepared in this buffer with a final DMSO concentration of 0.5%. The highest peptide concentration was 100 nM, which was diluted 6 times in 2-fold dilutions. SPR analysis was run at 25°C at a flow rate of 50 μl / min with an association time of 60 seconds and dissociation times of 400 to 1200 seconds depending on the individual peptides. Data were corrected for DMSO exclusion volume effects. All data were double-referenced to blank injections and reference surfaces using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software version 2.0c (BioLogic Software). A simple 1:1 binding model was used to fit the data to account for mass transfer effects where appropriate.

[1518] Certain peptide ligands of the present invention were tested in the above described nexin-4 binding assay and the results are shown in Table 1B and Table 2:

[1519] Table 1B: Direct binding data of selected peptide ligands of the present invention

[1520]

[1521] Where "A" represents L-alanine, and "a" represents L-alanine, * is the average of two experiments

[1522] Table 2: Competition binding data for selected peptide ligands of the present invention

[1523]

[1524]

[1525]

[1526]

[1527]

[1528]

[1529] Certain bicyclic peptides of the present invention were tested in the above-mentioned SPR assay and the results are shown in Table 3:

[1530] Table 3: SPR data of selected peptide ligands of the present invention

[1531]

[1532]

[1533] n = average number of trials

[1534] Certain bicyclic peptides of the present invention were conjugated to cytotoxic agents and tested in the above-mentioned SPR assay, with the results shown in Table 4:

[1535] Table 4: SPR data of BDC selected in the present invention

[1536]

[1537]

[1538] In vivo studies

[1539] In each of Examples 1 to 5 and 9, the following methods were used for each study:

[1540] Test and positive controls

[1541] Table 5-1

[1542] serial number Physical description Molecular weight purity Storage conditions BCY7683 freeze-dried powder 3852.59 99.6％ Store at -80°C BCY7825 freeze-dried powder 4122.85 98.40％ Store at -80°C BCY7826 freeze-dried powder 3821.83 99.70％ Store at -80°C BCY8234 freeze-dried powder 2954.34 98.1％ Store at -80°C BCY8242 freeze-dried powder 6346.46 97.40％ Store at -80°C BCY8245 freeze-dried powder 4173.85 99.60％ Store at -80°C BCY8253 freeze-dried powder 4218.96 97.00％ Store at -80°C BCY8254 freeze-dried powder 4213.96 99.40％ Store at -80°C BCY8255 freeze-dried powder 4214.97 99.40％ Store at -80°C BCY8549 freeze-dried powder 4157.81 95.70％ Store at -80°C BCY8550 freeze-dried powder 4228.98 99.20％ Store at -80°C BCY8781 freeze-dried powder 4173.85 99.0％ Store at -80°C BCY8783 freeze-dried powder 4209.91 96.80％ Store at -80°C BCY8784 freeze-dried powder 4245.96 95.70％ Store at -80°C

[1543] Experimental methods and procedures

[1544] (i) Observation

[1545] All procedures related to animal handling, care, and treatment in the studies were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec and followed the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). During routine monitoring, animals were examined daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain / loss, eye / hair gloss, and any other abnormal effects stated in the operating procedures. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[1546] (ii) Tumor measurements and endpoints

[1547] The primary endpoint was to see if tumor growth could be delayed or if the mice could be cured. Tumor volume was measured in two dimensions three times a week using calipers and expressed in mm using the following formula: 3 Indicates volume: V = 0.5a × b 2 , where a and b are the major and minor diameters of the tumor, respectively. Tumor dimensions were then used to calculate the T / C value. The T / C value (percentage) represents the antitumor effect; T and C are the mean volumes of the treatment and control groups, respectively, on a given day.

[1548] TGI was calculated for each group using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100;T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the day treatment started, and V i The vehicle control group was compared with T i The mean tumor volume on the same day, V0, is the mean tumor volume of the vehicle group on the day treatment started.

[1549] (iii) Statistical analysis

[1550] A statistical summary of tumor volume for each group at each time point is provided, including the mean and standard error of the mean (SEM).

[1551] The differences in tumor volume between the groups were statistically analyzed based on the data obtained at the optimal treatment time point after the final dose.

[1552] One-way ANOVA was performed to compare tumor volumes between groups, and when a significant F-statistic (ratio of treatment difference to error difference) was obtained, the Games-Howell test was used to compare between groups. All data were analyzed using GraphPadPrism 5.0. P < 0.05 was considered statistically significant.

[1553] Example 1: In vivo efficacy testing of BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254 and BCY8255 in treating NCI-H292 xenografts (a non-small cell lung cancer (NSCLC) model) in BALB / c nude mice.

[1554] 1. Research Objectives

[1555] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, and BCY8255 in treating the NCI-H292 xenograft model in BALB / c nude mice.

[1556] 2. Experimental Design

[1557] Table 5-2

[1558]

[1559]

[1560] Note: n: number of animals; Dosing volume: 10 μL / g based on body weight.

[1561] *From day 7 onwards, the dose of BCY7683 was reduced to 3 mg / kg.

[1562] ** Treatment schedules were adjusted based on body weight on the day of dosing.

[1563] 3. Materials

[1564] 3.1 Animals and housing conditions

[1565] 3.1.1. Animals

[1566] Species: House mouse

[1567] Strain: Balb / c nude mice

[1568] Age: 6-8 weeks

[1569] Gender: Female

[1570] Weight: 18-22 grams

[1571] Number of animals: 12 mice for BCY7683 testing, 21 mice for BCY7825 and BCY7826, 18 mice for BCY8245, BCY8253, BCY8254, and BCY8255, plus spares.

[1572] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[1573] 3.1.2. Rearing conditions

[1574] Mice were maintained in individually ventilated cages at constant temperature and humidity, with 3 animals per cage.

[1575] Temperature: 20-26°C

[1576] Humidity 40-70%.

[1577] Cage: Made of polycarbonate. Dimensions: 300 mm x 180 mm x 150 mm. Bedding: corn cobs, changed twice a week.

[1578] Diet: Animals had free access to irradiated dry pelleted food throughout the study.

[1579] Drinking water: Animals had free access to sterile drinking water.

[1580] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date.

[1581] Animal identification: tag animals using ear codes.

[1582] 3.2 Test and Positive Control

[1583] 4. Experimental Methods and Procedures

[1584] 4.1 Cell culture

[1585] NCI-H292 tumor cells were maintained in culture medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO2 in air. Tumor cells were routinely subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[1586] 4.2 Tumor inoculation

[1587] Each mouse was inoculated subcutaneously on the right flank with NCI-H292 tumor cells (10 × 106 ) for tumor growth. When the average tumor volume reached approximately 158–406 mm 3 At 4 hr, animals were randomized and treatment started. The experimental design table below shows the test article administration and the number of animals in each group.

[1588] 4.3 Preparation of test substance

[1589] Table 5-3

[1590]

[1591] Table 5-4

[1592]

[1593]

[1594] Table 5-5

[1595]

[1596] Table 5-6

[1597]

[1598]

[1599] 1. Histidine buffer: 25 mM histidine, pH 7, 10% sucrose

[1600] 2. Acetate buffer: 50 mM acetate / acetic acid, pH 5, 10% sucrose

[1601] 3. Divide BCY8245 (1 mg / mL), BCY8253 (1 mg / mL), and BCY8255 (1 mg / mL) stock solutions into separate tubes and store at -80°C.

[1602] 4.4 Sample collection

[1603] At the end of the study, plasma was collected 5, 15, 30, 60, and 120 minutes after the last dose.

[1604] 5. Results

[1605] 5.1 Tumor growth curve

[1606] Tumor growth curve Figures 1 to 10 shown.

[1607] 5.2 Tumor volume trajectory

[1608] The following table shows the average tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts:

[1609] Table 5-7: Trajectory of tumor volume changes over time

[1610]

[1611] Table 6: Tumor volume trajectory over time

[1612]

[1613] Table 7: Tumor volume trajectory over time

[1614]

[1615]

[1616] Table 8: Trajectory of tumor volume changes over time

[1617]

[1618] Table 9: Trajectory of tumor volume changes over time

[1619]

[1620]

[1621] 5.3 Tumor Growth Inhibition Analysis

[1622] Tumor growth inhibition rates of BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, and BCY8255 in the NCI-H292 xenograft model were calculated on day 14 based on tumor volume measurements.

[1623] Table 10: Tumor Growth Inhibition Analysis

[1624]

[1625] a. Mean ± SEM.

[1626] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1627] Table 11: Tumor Growth Inhibition Analysis

[1628]

[1629]

[1630] Table 12: Tumor Growth Inhibition Analysis

[1631]

[1632] a. Mean ± SEM.

[1633] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1634] Table 13-1: Tumor growth inhibition analysis

[1635]

[1636]

[1637] a. Mean ± SEM.

[1638] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1639] 6. Summary and Discussion of Results

[1640] In this study, the therapeutic efficacy of BCY7683, BCY7825, BCY7826, BCY8245, BCY8253, BCY8254, and BCY8255 was evaluated in the NCI-H292 xenograft model. Figures 1 to 10 and Tables 5-7 to 13-1 show the measured tumor volumes of all treatment groups at various time points.

[1641] On day 14, the mean tumor size in vehicle-treated mice reached 879 mm 3 BCY7683 at 5 mg / kg showed rapid tumor regression, but the treatment induced severe weight loss, so the dose was suspended on the third day and adjusted to 3 mg / kg on the seventh day. Finally, BCY7683 (TV = 47 mm 3 , TGI=118.4%, p<0.001) produced significant anti-tumor efficacy, and all mice survived to the endpoint.

[1642] On day 21, the average tumor size of vehicle-treated mice reached 1107 mm 3 BCY7825 was administered at 1 mg / kg (TV = 1063 mm 3 , TGI = 4.3%, p > 0.05) did not produce any anti-tumor activity, BCY7825 at 3 mg / kg biw (TV = 80 mm 3, TGI=108.3%,p<0.001) and 3mg / kg qw (TV=177mm 3 , TGI = 97.9%, p < 0.001) caused rapid tumor regression and showed strong anti-tumor activity.

[1643] BCY7826 (1 mg / kg) (TV = 774 mm 3 , TGI = 34.9%, p > 0.05) did not produce significant anti-tumor activity, BCY7826 (3 mg / kg biw) (TV = 93 mm 3 , TGI=106.9%,p<0.001) and 3mg / kg (TV=425mm 3 , TGI = 71.7%, p < 0.001) qw produced a frequency-dependent antitumor activity. Among them, BCY7826 (3 mg / kg biw) induced significant tumor regression.

[1644] BCY8245, BCY8253, BCY8254, and BCY8255 did not produce significant antitumor activity at 1 mg / kg. After increasing the dose to 3 mg / kg starting on day 7, all four test substances showed significant antitumor activity, but the efficacy was not further improved when the dose was increased to 5 mg / kg on day 21. In this study, all treated animals showed sustained weight loss during the dosing regimen, which may be due to tumor burden and toxicity of the test substances.

[1645] BCY8245, 3mg / kg,qw (TV=149mm 3 ,TGI=101.4%, p<0.001), 3mg / kg,biw(TV=65mm 3 , TGI=112.2%, p<0.001) and 5mg / kg,qw(TV=83mm 3 , TGI = 109.8%, p < 0.001) produced significant anti-tumor activity.

[1646] BCY8253, 3mg / kg, qw (TV = 98mm 3 ,TGI=108.1%, p<0.001), 3mg / kg,biw(TV=49mm 3 , TGI=114.3%, p<0.001) and 5mg / kg,qw(TV=68mm 3 , TGI = 111.9%, p < 0.001) produced significant anti-tumor activity.

[1647] BCY8255, 3mg / kg,qw (TV=100mm 3,TGI=107.9%, p<0.001), 3mg / kg,biw(TV=96mm 3 , TGI=108.5%, p<0.001) and 5mg / kg,qw(TV=97mm 3 , TGI = 108.5%, p < 0.001) produced significant anti-tumor activity.

[1648] All of these test substances showed comparable antitumor activity at 3 mg / kg qw, 3 mg / kg biw, and 5 mg / kg qw, with no further improvement in efficacy when the dose or dosing frequency was increased.

[1649] In this study, animals treated with BCY8253 5 mg / kg showed an average body weight loss of more than 15% on day 9, while mice in other groups maintained their body weight well.

[1650] Example 2: In vivo efficacy testing of BCY7825, BCY8245, BCY8253, BCY8254, and BCY8255 in treating HT-1376 xenografts (bladder cancer model) in CB17-SCID mice

[1651] 1. Research Objectives

[1652] The purpose of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of HT-1376 xenografts in CB17-SCID mice.

[1653] 2. Experimental Design

[1654] Table 13-2

[1655]

[1656] a 1 mg / kg for the first week, then 3 mg / kg for the next 2 weeks

[1657] 3. Materials

[1658] 3.1 Animals and housing conditions

[1659] 3.1.1. Animals

[1660] Species: House mouse

[1661] Germline: CB17-SCID

[1662] Age: 6-8 weeks

[1663] Gender: Female

[1664] Weight: 18-22 grams

[1665] Number of animals: 21-41 mice plus spare

[1666] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[1667] 3.1.2. Rearing conditions

[1668] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1669] Temperature: 20-26°C.

[1670] Humidity 40-70%.

[1671] Cage: Made of polycarbonate. Dimensions: 300 mm x 180 mm x 150 mm. Bedding: corn cobs, changed twice a week.

[1672] Diet: Animals had free access to irradiated dry pelleted food throughout the study.

[1673] Drinking water: Animals had free access to sterile drinking water.

[1674] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date.

[1675] Animal identification: tag animals using ear codes.

[1676] 4. Experimental Methods and Procedures

[1677] 4.1 Cell culture

[1678] HT-1376 tumor cells were maintained in vitro as monolayers in EMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO₂ in air. Tumor cells were routinely passaged twice weekly using trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[1679] 4.2 Tumor inoculation

[1680] Each mouse was inoculated subcutaneously on the right flank with 0.2 ml of HT-1376 tumor cells (5 × 10 6 ) for tumor growth. When the average tumor volume reached 153-164 mm 3 The animals were randomized at 4 hr. The experimental design table below shows the test article administration and the number of animals in each group.

[1681] 4.3 Preparation of test substance

[1682] Table 13-3

[1683]

[1684]

[1685] Table 13-4

[1686]

[1687] 4.4 Sample collection

[1688] At the end of the study, plasma was collected at 5, 15, 30, 60, and 120 minutes after the last dose for Groups 3, 4, 5, 6, and 7. Plasma was collected at 5, 15, 30, 60, and 120 minutes after the last dose for Groups 11, 14, and 17. Tumors were collected 2 hours after the last dose for Groups 11, 14, and 17. Tumors were collected 2 hours after the last dose for Groups 8, 9, 10, 12, 13, 15, and 16.

[1689] 5. Results

[1690] 5.1 Tumor growth curve

[1691] Tumor growth curve Figures 11 to 18 shown.

[1692] 5.2 Tumor volume trajectory

[1693] Tables 14 and 15 show the changes in mean tumor volume over time in female CB17-SCID mice bearing HT-1376 xenografts.

[1694]

[1695] Table 15: Tumor volume changes over time

[1696]

[1697] 5.3 Tumor Growth Inhibition Analysis

[1698] Tumor growth inhibition rates of the test articles in the HT-1376 xenograft model were calculated based on tumor volume measurements on day 21 after the start of treatment.

[1699] Table 16: Tumor Growth Inhibition Analysis

[1700]

[1701]

[1702] a. Mean ± SEM.

[1703] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1704] Table 17-1: Tumor Growth Inhibition Analysis

[1705]

[1706] a. Mean ± SEM.

[1707] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1708] 6. Summary and Discussion of Results

[1709] Groups 1-7

[1710] In this study, the therapeutic efficacy of the test article was evaluated in the HT-1376 xenograft model. Figures 11 to 15 and Tables 14 and 15 show the measured tumor volumes for all treatment groups at various time points.

[1711] On day 21, the mean tumor size in vehicle-treated mice reached 884 mm 3 BCY7825 (1 mg / kg) (TV = 772 mm 3 , TGI=15.2%, p>0.05) did not produce significant antitumor activity, BCY7825 (3mg / kg) (TV=545mm 3 , TGI = 46.9%, p < 0.05) produced significant anti-tumor activity.

[1712] BCY8245, BCY8253, BCY8254, and BCY8255 at 1 mg / kg produced mild antitumor activity, and better efficacy was observed after increasing the dose to 3 mg / kg from day 7 onwards.

[1713] In this study, some mice treated with 3 mg / kg of the test substance showed more than 10% weight loss, one mouse treated with 3 mg / kg of BCY8255 was found dead on day 16, and mice in the vehicle and 1 mg / kg of BCY7825 groups maintained good body weight.

[1714] Groups 8-17

[1715] In this study, the therapeutic efficacy of the test article was evaluated in the HT-1376 xenograft model. Figures 16 to 18 and Table 16 and Table 17-1 show the measured tumor volumes of all treatment groups at various time points.

[1716] BCY8245, 3mg / kg,qw (TV=603mm 3 ,TGI=50.9%, p<0.01),3mg / kg,biw

[1717] (TV=407mm 3 , TGI=72.3%, p<0.001) and 5mg / kg,qw(TV=465mm 3 , TGI = 66.0%, p < 0.001) produced significant anti-tumor activity.

[1718] BCY8253, 3mg / kg,qw (TV=533mm 3 ,TGI=58.5%, p<0.01),3mg / kg,biw

[1719] (TV=442mm 3 , TGI=68.4%, p<0.001) and 5mg / kg, qw (TV=442mm 3 , TGI = 68.5%, p < 0.001) produced significant anti-tumor activity.

[1720] BCY8255, 3mg / kg,qw (TV=538mm 3 ,TGI=58.0%, p<0.01),3mg / kg,biw

[1721] (TV=390mm 3 , TGI=74.1%, p<0.001) and 5mg / kg,qw(TV=516mm 3 , TGI = 60.3%, p < 0.001) produced significant anti-tumor activity.

[1722] In this study, 5 mg / kg qw of BCY8245 and BCY8253 caused greater than 10% body weight loss in animals over the treatment period.

[1723] Example 3: In vivo efficacy study of BCY8245, BCY8253, and BCY8255 in treating Panc2.13 xenografts (pancreatic cancer model) in Balb / c nude mice

[1724] 1. Research Objectives

[1725] The purpose of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of Panc2.13 xenografts in Balb / c nude mice.

[1726] 2. Experimental Design

[1727] Table 17-2

[1728]

[1729] 3. Materials

[1730] 3.1 Animals and housing conditions

[1731] 3.1.1 Animals

[1732] Species: House mouse

[1733] Strain: Balb / c nude mice

[1734] Age: 6-8 weeks

[1735] Gender: Female

[1736] Weight: 18-22 grams

[1737] Number of animals: 41 mice plus spare

[1738] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[1739] 3.1.2. Rearing conditions

[1740] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1741] Temperature: 20-26°C.

[1742] Humidity 40-70%.

[1743] Cage: Made of polycarbonate. Dimensions: 300 mm x 180 mm x 150 mm. Bedding: corn cobs, changed twice a week.

[1744] Diet: Animals had free access to irradiated dry pelleted food throughout the study.

[1745] Drinking water: Animals had free access to sterile drinking water.

[1746] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date.

[1747] Animal identification: tagging animals using ear codes.

[1748] 4. Experimental Methods and Procedures

[1749] 4.1 Cell culture

[1750] Panc2.13 tumor cells were maintained in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units / ml of human recombinant insulin at 37°C in an atmosphere containing 5% CO₂ in air. Tumor cells were routinely subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[1751] 4.2 Tumor inoculation

[1752] Each mouse was inoculated subcutaneously on the right flank with Panc2.13 tumor cells (5 × 10 6 ) for tumor formation. When the average tumor volume reached 149 mm 3 41 animals were randomized. The experimental design table shows the test article administration and the number of animals in each group.

[1753] 4.3 Preparation of test substance

[1754] Table 17-3

[1755]

[1756]

[1757] 4.4 Sample collection

[1758] At the end of the study, tumors from all groups were collected 2 h after the last dose.

[1759] 5. Results

[1760] 5.1 Tumor growth curve

[1761] Tumor growth curve Figures 19 to 21 shown.

[1762] 5.2 Tumor volume trajectory

[1763] Table 18 below shows the changes in mean tumor volume over time in female Balb / c nude mice bearing Panc2.13 xenografts.

[1764] Table 18: Trajectory of tumor volume changes over time

[1765]

[1766]

[1767] 5.3 Tumor Growth Inhibition Analysis

[1768] The tumor growth inhibition rate of the test articles in the Panc2.13 xenograft model was calculated based on tumor volume measurements on day 14 after the start of treatment.

[1769] Table 19-1: Tumor Growth Inhibition Analysis

[1770]

[1771] a. Mean ± SEM.

[1772] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1773] 6. Summary and Discussion of Results

[1774] In this study, the therapeutic efficacy of the test article was evaluated in the Panc2.13 xenograft model. Figures 19 to 21 and Table 18 and Table 19-1 show the measured tumor volumes of all treatment groups at various time points.

[1775] BCY8245, 3mg / kg,qw (TV=271mm 3 ,TGI=69.2%, p<0.01),3mg / kg,biw(TV=231mm 3 , TGI=79.1%, p<0.001) and 5mg / kg,qw(TV=238mm 3 , TGI = 77.5%, p < 0.001) produced significant anti-tumor activity.

[1776] BCY8253, 3mg / kg,qw (TV=324mm 3 ,TGI=59.8%, p<0.01),3mg / kg,biw(TV=219mm 3 , TGI=82.2%, p<0.001) and 5mg / kg,qw(TV=170mm 3 , TGI = 94.5%, p < 0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1777] BCY8255, 3mg / kg,qw (TV=310mm 3 ,TGI=59.5%, p<0.01),3mg / kg,biw(TV=251mm 3, TGI=74.3%, p<0.001) and 5mg / kg,qw(TV=209mm 3 , TGI = 85.1%, p < 0.001) produced significant anti-tumor activity.

[1778] In this study, animals in all 5 mg / kg qw groups lost an average of more than 15% body weight, particularly those in the BCY8253 and BCY8255 5 mg / kg groups, which lost more than 20% body weight during the treatment period.

[1779] Example 4: In vivo efficacy study of BCY8245, BCY8253 and BCY8255 in the treatment of MDA-MB-468 xenografts (breast cancer model) in Balb / c nude mice

[1780] 1. Research Objectives

[1781] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8245, BCY8253, and BCY8255 in the treatment of MDA-MB-468 xenografts in Balb / c nude mice.

[1782] 2. Experimental Design

[1783] Table 19-2

[1784]

[1785] 3. Materials

[1786] 3.1 Animals and housing conditions

[1787] 3.1.1 Animals

[1788] Species: House mouse

[1789] Strain: Balb / c nude mice

[1790] Age: 6-8 weeks

[1791] Gender: Female

[1792] Weight: 18-22 grams

[1793] Number of animals: 41 mice plus spare

[1794] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[1795] 3.1.2. Rearing conditions

[1796] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1797] Temperature: 20-26°C.

[1798] Humidity 40-70%.

[1799] Cage: Made of polycarbonate. Dimensions: 300 mm x 180 mm x 150 mm. Bedding: corn cobs, changed twice a week.

[1800] Diet: Animals had free access to irradiated dry pelleted food throughout the study.

[1801] Water: Animals had free access to sterile drinking water.

[1802] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date.

[1803] Animal identification: tagging animals using ear codes.

[1804] 4. Experimental methods and procedures

[1805] 4.1 Cell culture

[1806] Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% carbon dioxide in air. Tumor cells were routinely subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[1807] 4.2 Tumor inoculation

[1808] Each mouse was subcutaneously inoculated with 0.2 ml of MDA-MB-468 tumor cells (10 × 10 6 ) for tumor formation, PBS was supplemented with 50% Matrigel. When the average tumor volume reached 196 mm 3 41 animals were randomized. The experimental design table shows the test article administration and the number of animals in each group.

[1809] 4.3 Preparation of test substance

[1810] Table 19-3

[1811]

[1812]

[1813] 1. Histidine buffer: 25 mM histidine, pH 7, 10% sucrose

[1814] 2. Acetate buffer: 50 mM acetate / acetic acid, pH 5, 10% sucrose

[1815] 3. Divide BCY8245 (1 mg / mL), BCY8253 (1 mg / mL), and BCY8255 (1 mg / mL) stock solutions into separate tubes and store at -80°C.

[1816] 4.4 Sample collection

[1817] On study day 21, plasma was collected from Groups 2, 5, and 8 at 5, 15, 30, 60, and 120 minutes after the last dose. Tumors were collected 2 hours after the last dose from Groups 1, 3, 6, and 9. Animals from Groups 4, 7, and 10 were maintained for another 21 days without any dosing, and tumors from these groups were collected on day 42.

[1818] 5. Results

[1819] 5.1 Tumor growth curve

[1820] Tumor growth curve Figures 22 to 24 shown.

[1821] 5.2 Tumor volume trajectory

[1822] Tables 20 and 21 below show the changes in mean tumor volume over time in female Balb / c nude mice bearing MDA-MB-468 xenografts.

[1823]

[1824]

[1825] 5.3 Tumor Growth Inhibition Analysis

[1826] The tumor growth inhibition rate of the test articles in the MDA-MB-468 xenograft model was calculated based on the tumor volume measurements on day 21 after the start of treatment.

[1827] Table 22-1: Tumor Growth Inhibition Analysis

[1828]

[1829] a. Mean ± SEM.

[1830] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1831] 6. Summary and Discussion of Results

[1832] In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. Figures 22 to 24 and Tables 20 to 22-1 show the measured tumor volumes of all treatment groups at various time points.

[1833] BCY8245, 3mg / kg, qw (TV=85mm 3 ,TGI=144.2%, p<0.001),3mg / kg,biw(TV=22mm 3 , TGI=169.8%, p<0.001) and 5mg / kg,qw(TV=29mm 3 , TGI=168.4%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1834] BCY8253, 3mg / kg, qw (TV=109mm 3 ,TGI=134.7%, p<0.001),3mg / kg,biw(TV=29mm 3 , TGI=168.3%, p<0.001) and 5mg / kg,qw(TV=40mm 3 , TGI=163.9%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1835] BCY8255, 3mg / kg, qw (TV=136mm 3 ,TGI=125.1%, p<0.001),3mg / kg,biw(TV=31mm 3 , TGI=166.8%, p<0.001) and 5mg / kg, qw (TV=42mm 3 , TGI=161.3%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1836] From day 21, dosing of the 5 mg / kg group was suspended, and no tumor recurrence was observed during an additional 3-week monitoring period.

[1837] In this study, BCY8253 and BCY8255 5 mg / kg caused severe weight loss in animals, with mouse 10-1 in the BCY8253 5 mg / kg group dying on day 20.

[1838] Example 5: In vivo efficacy testing of BCY8549, BCY8550, BCY8783 and BCY8784 in treating NCI-H292 xenografts (a non-small cell lung cancer (NSCLC) model) in BALB / c nude mice.

[1839] 1. Research Objectives

[1840] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8549, BCY8550, BCY8783, and BCY8784 in the treatment of NCI-H292 xenografts in Balb / c nude mice.

[1841] 2. Experimental Design

[1842] Table 22-2

[1843]

[1844] 3. Materials

[1845] 3.1 Animals and housing conditions

[1846] 3.1.1. Animals

[1847] Species: House mouse

[1848] Strain: Balb / c nude mice

[1849] Age: 6-8 weeks

[1850] Gender: Female

[1851] Weight: 18-22 grams

[1852] Number of animals: 43 mice plus spare

[1853] Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co. Ltd

[1854] 3.1.2. Rearing conditions

[1855] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 4 animals per cage.

[1856] Temperature: 20-26°C.

[1857] Humidity 40-70%.

[1858] Cage: Made of polycarbonate. Dimensions: 300 mm x 180 mm x 150 mm. Bedding: corn cobs, changed twice a week.

[1859] Diet: Animals had free access to irradiated dry pelleted food throughout the study.

[1860] Water: Animals had free access to sterile drinking water.

[1861] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date.

[1862] Animal identification: tagging animals using ear codes.

[1863] 4. Experimental methods and procedures

[1864] 4.1 Cell culture

[1865] NCI-H292 tumor cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO₂. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[1866] 4.2 Tumor inoculation

[1867] Each mouse was inoculated subcutaneously on the right flank with NCI-H292 tumor cells (10 × 10 6 ) for tumor formation. When the average tumor volume reached 168mm 3 At 43 animals were randomized. The experimental design table shows the test article administration and the number of animals in each group.

[1868] Preparation of test substance

[1869] Table 22-3

[1870]

[1871] 4.4 Sample collection

[1872] At the end of the study, plasma was collected from mice in groups 2 and 3 at 5 min, 15 min, 30 min, 1 h, and 2 h after the last dose.

[1873] 5. Results

[1874] 5.1 Tumor growth curve

[1875] Tumor growth curve Figures 25 to 28 shown.

[1876] 5.2 Tumor volume trajectory

[1877] Table 23 below shows the changes in mean tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts.

[1878] Table 23: Trajectory of tumor volume changes over time

[1879]

[1880] 5.3 Tumor Growth Inhibition Analysis

[1881] Tumor growth inhibition rates of the test articles in the NCI-H292 xenograft model were calculated based on tumor volume measurements on day 14 after the start of treatment.

[1882] Table 24: Tumor Growth Inhibition Analysis

[1883]

[1884] a. Mean ± SEM.

[1885] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1886] 6. Summary and Discussion of Results

[1887] In this study, the therapeutic efficacy of BCY was evaluated in the NCI-H292 xenograft model. Figures 25 to 28 and Tables 23 and 24 show the measured tumor volumes for all treatment groups at various time points.

[1888] On day 14, the mean tumor size in vehicle-treated mice reached 843 mm 3 While BCY8550 at 3 mg / kg did not show significant antitumor activity, the other compounds at 3 mg / kg did exhibit significant antitumor activity. Among them, BCY8783 and BCY8784 exhibited comparable tumor-suppressing effects to BCY8245 and effectively regressed tumors.

[1889] All mice in the study maintained a good body weight.

[1890] Example 6: Correlation between copy number variation (CNV) and gene expression of integrin-4 in various tumor types

[1891] method

[1892] 1. In cBioPortal( http: / / www.cbioportal.org / ) and search for NECTIN4.

[1893] (a) Delete the temporary study.

[1894] (b) Deselect studies with overlapping samples to prevent sample bias (based on warnings in cBioPortal) - always keep the PanCancer study if it can be selected.

[1895] (c) Studies selected for analysis (Table 25).

[1896] Table 25: Studies analyzed from cBioPortal and research institutions

[1897]

[1898]

[1899]

[1900] 2. Export CNV and RNA expression data from cBioPortal.

[1901] 3. Test whether CNV is statistically significantly associated with changes in integrin-4 mRNA expression (log2 not applied).

[1902] (a) Non-parametric Kruskal-Wallis test (significance threshold: p < 0.01) was run in GraphPad Prism (7.04) and R / R workbench (studio).

[1903] (i) GraphPad Prism: Set up the list and run nonparametric tests that are unmatched or unpaired and do not use a Gaussian distribution.

[1904] (ii) Software packages used in R:

[1905] 1.XLConnect

[1906] 2. dplyr

[1907] 3.Kruskal-Wallis rank sum test: Kruskal test.

[1908] 4. Dunn's test was used in the R / R workbench to adjust for multiple comparisons (including all possible comparisons even if n=1 in a group) (significance threshold: p<0.025).

[1909] (a) dunn.test = "bonferonni" using the multiple comparison method.

[1910] result

[1911] The results are shown in Table 26 below. Among the 41 publicly available TCGA datasets reporting tumor CNV and mRNA gene expression data for integrin-4, many indications have reported cases with integrin-4 copy number gain (2-3 copies) or amplification (>3 copies). In addition, isolated cases with shallow deletions (<2 copies) have been demonstrated, and tumors with deep deletions have rarely been reported, consistent with loss of more than 1 copy or biallelic integrin-4 loss. The indications in which amplification was most frequently detected were breast cancer (10-22%), bladder cancer (20%), lung cancer (5-7%), and hepatocellular carcinoma (8%). The indications in which copy number loss was most frequent were renal chromophobe carcinoma (77%), renal clear cell carcinoma (RCC) (6.5%), sarcoma (10%), colon cancer (10%), head and neck cancer (7%), and squamous cell carcinoma of the lung. These data indicate that there is a range of CNVs within and across tumor indications, and that there is diversity in copy number patterns across different indications. In addition to the CNVs within the TCGA dataset, the median expression level of integrin-4 mRNA for each indication spans approximately 2 10 Therefore, given the range of integrin-4 mRNA expression levels and the CNVs observed between and among tumor types, statistical tests were performed to identify potential correlations between integrin-4 mRNA levels and integrin-4 CNVs in each TCGA dataset / indication. Tumors for each indication were classified into 1 of 5 categories:

[1912] a) Deep deletion;

[1913] b) shallow deletion;

[1914] c) diploid;

[1915] d) increase; or

[1916] e) Amplification.

[1917] A Kruskall-Wallis test was then performed to detect whether the distribution of mRNA expression values ​​for each category differed between the categories (P < 0.01). For those TCGA datasets with P < 0.01, post hoc tests were performed to identify which categories differed from each other by calculating the Z-statistic and calculating the adjusted P value (Bonferonni). To simplify interpretation, pairwise comparisons relative to diploid were performed for each indication (although P values ​​were calculated for all pairs). 18 / 41 TCGA studies achieved Kruskall-Wallis P < 0.01 and Bonferonni P < 0.025 in the increase relative diploid and / or amplification relative diploid comparisons, indicating that increased integrin-4 mRNA expression was associated with increased integrin-4 copy number. These 18 studies represented 14 independent tumor histologies:

[1918] Breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC), and gastric cancer.

[1919] Furthermore, six studies had reduced mRNA expression associated with copy number loss. Four of these six studies not only showed an association between CNV loss and reduced expression, but also reported that CNV gain was associated with higher expression:

[1920] Gastric cancer, squamous cell lung cancer, colon cancer and thyroid cancer.

[1921] Two indications, renal chromophobe carcinoma and prostate cancer, have only been reported to be associated with CNV loss and low transcript abundance. In addition, a separate study in prostate cancer (Metastatic Prostate Cancer, SU2C / PCF Dream Team (Robinson et al., Cell 2015)) showed that copy number gain was associated with high expression (relative to diploidy).

[1922] These observed tumor CNV losses and gains with mRNA expression levels may represent the mechanism behind integrins-4 tumor protein expression in those indications where such correlations are observed. Clearly, there are indications (e.g., hepatocellular carcinoma) where CNVs do not appear to affect mRNA expression levels in a predictable pattern. The in vivo preclinical efficacy of certain integrins-4 bicyclic drug conjugates of the present invention has been shown to correlate with integrins-4 protein expression as measured by IHC. Therefore, if tumor integrins-4 CNVs correlate with mRNA levels and predict protein expression levels, then patients with tumors that formally may have copy number increases (gains or amplifications) are more likely to respond to the integrins-4 bicyclic drug conjugates of the present invention. If patients with increased CNVs in integrins-4 can be identified, then this information can be used to select patients for treatment with the integrins-4 bicyclic drug conjugates of the present invention.

[1923]

[1924]

[1925]

[1926]

[1927]

[1928]

[1929]

[1930]

[1931]

[1932]

[1933] Example 7: Expression analysis of integrin-4 in 6 cell lines

[1934] 1. Research Objectives

[1935] The aim of the study was to evaluate the expression of integrin-4 by flow cytometry in six cell lines, including two breast cancer (T-47D, MDA-MB-468), three lung cancer (NCI-H292, NCI-H322, NCI-H526) and one fibrosarcoma (HT-1080) cell lines.

[1936] 2. Group Design

[1937] Panel for FCM in T-47D, MDA-MB-468, NCI-H292, NCI-H322, and HT-1080

[1938]

[1939] NCI-H526 group

[1940]

[1941] 3. Materials

[1942] 3.1 Sample

[1943] Cell Series List

[1944] Table 27-1

[1945]

[1946] Reagents

[1947] Antibodies and kits for flow cytometry analysis

[1948] Table 27-2

[1949]

[1950] DPBS (Corning-21-031-CV)

[1951] Staining buffer (eBioscience-00-4222)

[1952] Fixation Buffer (BD-554655)

[1953] 3.3. Instruments

[1954] Eppendorf Centrifuge 5810R

[1955] BD FACS Canto flow cytometer (BD)

[1956] 4. Experimental methods and procedures

[1957] 4.1 Sample collection

[1958] Collect cell lines in the exponential growth phase. Count cells using a hemocytometer using trypan blue staining. Centrifuge cells at 400 × g for 5 minutes at 4°C, wash cells twice with staining buffer, and then resuspend cells to 1 × 10 cells / mL in staining buffer. 7 / mL.

[1959] 4.2 Antibody staining

[1960] 1) Aliquot 100 μL of cell suspension into each well of a 96-well V-plate.

[1961] 2) Add isotype control or antibody to the suspended cells and incubate at 4°C in the dark for 30 minutes.

[1962] 3) Wash the cells 2× by centrifugation at 400×g for 5 minutes at 4°C and discard the supernatant.

[1963] 4) Resuspend the cells with 100 μL of fixation buffer and incubate at 4°C in the dark for 30 minutes.

[1964] 5) Wash the cells 2× by centrifugation at 300×g for 5 minutes at 4°C and remove the supernatant.

[1965] 6) Resuspend cells in 400 μL staining buffer.

[1966] 7) FACS data were analyzed using FlowJo V10 software.

[1967] 4.3 Data Analysis

[1968] All FACS data were analyzed using FlowJo V10 software and Graphpad Prism or Excel software.

[1969] 5. Results

[1970] 5.1 Group Gate Strategy

[1971] exist Figures 29-32 Gating strategy for nephrin-4 is shown in

[1972] 5.2 Data Analysis

[1973] 5.2.1. Cell line viability

[1974] The viability of the cell lines is as follows.

[1975] Table 27-3

[1976]

[1977]

[1978] 5.2.2. Positive expression of integrin-4 in cell lines

[1979] The positive expression and MFI of integrin-4 in 6 cell lines are shown in the table

[1980] Table 27-4

[1981] serial number cell lines Integrin-4 MFI-isotype MFI-Group 1 T-47D 99.0％ 132 1808 2 MDA-MB-468 99.0％ 184 2324 3 NCI-H292 97.9％ 180 729 4 NCI-H322 99.1％ 145 1655 5 NCI-H526 0.21％ 104 91.3 6 HT1080 1.53％ 134 134

[1982] 6. Discussion

[1983] Integrin-4 was highly expressed in breast cancer T-47D (99.0%), MDA-MB-468 (99.0%) and lung cancer NCI-H292 (97.9%), NCI-H322 (99.1%). Integrin-4 was not expressed in NCI-H526 and HT-1080.

[1984] Example 8: Analysis of Integrin-4 Expression in 9 CDX Cell Lines by Flow Cytometry

[1985] 1. Research Objectives

[1986] The aim of this project was to evaluate the surface expression of PVRL-4 in nine cell lines, including one breast cancer (MDA-MB-468), four lung cancer (NCI-H292, NCI-H358, NCI-H526, A549), one pancreatic cancer (Panc02.13), two colorectal cancer (HCT-116, HT-29) and one bladder cancer (HT1376) cell line.

[1987] 2. Group Design

[1988] A panel of FCMs in nine cell lines

[1989] Table 27-5

[1990] Fluorescent dyes blank Isotype group PE - Isotype control IgG2b Integrin-4 BV421 Live / Dead Live / Dead Live / Dead

[1991] 3. Materials

[1992] 3.1 Sample

[1993] Cell Series List

[1994] Table 27-6

[1995]

[1996] Reagents

[1997] 1) DPBS (Corning, 21-031-CV)

[1998] 2) Trypsin 0.25% (Invitrogen-25200072)

[1999] 3) Staining buffer (eBioscience, 00-4222)

[2000] 4) Fixation buffer (BD, 554655)

[2001] 5) Antibodies

[2002] Table 27-7

[2003] Fluorescence markers Catalog Number supplier illustrate PE Integrin-4 FAB2659P R&D AAAO0217021 PE Mouse IgG2b IC0041P R&D ...

Claims

1. Use of one or more reagents for determining the copy number of integrins-4 in the preparation of a kit for identifying or selecting a cancer patient for treatment with an integrins-4-targeted drug conjugate, wherein the identifying or selecting comprises: i) determining the copy number of integrin-4 in the patient's tumor sample; and ii) when the patient is identified as having more than two copies of Integrin-4, then identifying or selecting the patient for treatment with an Integrin-4-targeted drug conjugate.

2. Use of one or more reagents for determining the copy number of integrins-4 in the preparation of a kit for identifying or selecting cancer patients for treatment with an integrins-4-targeted drug conjugate, wherein the identification or selection comprises: More than two copies of Integrin-4 were identified in the patient tumor sample.

3. Use of one or more reagents for determining the number of copies of integrins-4 in the preparation of a kit for selecting a patient suffering from a cancer associated with increased integrins-4 gene expression for treatment, wherein the selection comprises: i) determining the copy number of integrin-4 in the patient's tumor sample; ii) identify patients with more than two copies of integrin-4; and iii) Select patients with more than two copies of integrin-4 for treatment.

4. The use according to any one of claims 1 to 3, wherein the drug conjugate comprises a peptide ligand.

5. The use according to claim 4, wherein the peptide ligand comprises: (a) a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and (b) a molecular scaffold that forms covalent bonds with at least three cystine residues of the polypeptide, such that at least two polypeptide rings are formed on the molecular scaffold.

6. The use according to claim 4 or 5, wherein the drug conjugate further comprises a cytotoxic agent.

7. The use according to claim 6, wherein the drug conjugate is BCY8245.

8. The use according to any one of claims 1 to 3, wherein the drug conjugate is an antibody drug conjugate.

9. The use according to claim 8, wherein the antibody drug conjugate comprises a cytotoxic agent.

10. The use according to claim 9, wherein the cytotoxic agent is monomethyl auristatin E.

11. The use according to any one of claims 8 to 10, wherein the antibody drug conjugate is Enfortumab Vedotin.

12. The method of any one of claims 1 to 11, wherein the cancer is selected from breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate cancer and gastric cancer.

13. The use according to claim 12, wherein the cancer is bladder cancer.

14. The use according to any one of claims 1 to 13, wherein the use comprises detecting the copy number of integrin-4 in the patient's tumor sample.

15. The use according to any one of claims 1 to 14, wherein the patient tumor sample is a tumor tissue sample or a blood sample.

16. Use of a bindin-4 targeted drug conjugate in the preparation of a drug for preventing, inhibiting or treating cancer in a patient, wherein the prevention, inhibition or treatment comprises administering a bindin-4-targeted drug conjugate to the patient, wherein the patient is identified as having more than two copies of bindin-4 in the tumor sample.

17. Use of a bindin-4 targeted drug conjugate in the preparation of a drug for preventing, inhibiting or treating cancer in a patient, wherein the prevention, inhibition or treatment comprises: i) determining the copy number of integrin-4 in the patient's tumor sample; and ii) when the patient is identified as having more than two copies of Integrin-4 in the tumor sample, administering an Integrin-4-targeting drug conjugate to the patient.

18. Use of a bindin-4 targeted drug conjugate in the preparation of a medicament for treating, preventing or inhibiting cancer in a subject, wherein the cancer is characterized by having more than two copies of bindin-4 in a tumor sample.

19. Use of a bindin-4 targeted drug conjugate in the preparation of a drug for preventing, inhibiting or treating cancer in a patient, wherein the prevention, inhibition or treatment comprises: The integrin-4 targeted drug conjugate is administered to a patient who has been diagnosed as having more than two copies of integrin-4 in a tumor sample.

20. The use according to any one of claims 16 to 19, wherein the drug conjugate comprises a peptide ligand comprising (a) a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and (b) a molecular scaffold that forms covalent bonds with at least three cystine residues of the polypeptide, such that at least two polypeptide rings are formed on the molecular scaffold.

21. The use according to claim 20, wherein the drug conjugate is BCY8245.

22. The use according to any one of claims 1 to 19, wherein the drug conjugate is an antibody drug conjugate.

23. The use according to claim 22, wherein the antibody drug conjugate comprises monomethyl auristatin E.

24. The use according to claim 22 or 23, wherein the antibody drug conjugate is Enfortumab Vedotin.

25. The use according to any one of claims 16 to 19, wherein: (a) With respect to claim 16-17 or 19, the patient tumor sample is a tumor tissue sample or a blood sample; and (b) With respect to claim 18, the sample from the subject's tumor is a tumor tissue sample or a blood sample.

26. The use according to any one of claims 16 to 24, wherein the cancer is selected from breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate cancer and gastric cancer.

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