Drug conjugate compounds for stimulating anti-tumor immune responses

Abstract

Despite important advances in cancer treatment due to the development of immune checkpoint inhibitors (ICIs), some cancers exhibit resistance to these agents. Because certain tumors are considered immunologically cold tumors and typically respond poorly to cancer immunotherapies, such as immune checkpoint blockade (ICB) therapy, there is a need to develop new approaches for the treatment of such cancers. The present application discloses methods and uses for enhancing anti-tumor immune responses in subjects suffering from sortilin-expressing cancers and / or treating subjects suffering from sortilin-expressing cancers, such as cancers resistant to immunotherapy, through the administration of conjugate compounds comprising a sortilin-targeting agent linked to an anticancer drug. Combination therapies comprising the conjugate compounds and immunotherapies, such as ICB, are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,561, filed November 14, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The Sequence Listing is submitted herewith as an XML file entitled G11718-00471-SSS_Seq listing.xml, created on November 14, 2023, and having a size of approximately 100 kilobytes. The contents of the foregoing file are incorporated herein by reference in their entirety.

[0003] The present invention relates generally to the field of oncology, and more specifically to the treatment of cancer. [Background technology]

[0004] The role of cancer in morbidity and mortality in human and animal populations means that there is a continuing need for new drugs that are effective against tumors. Eliminating tumors or reducing their size, or reducing the number of cancer cells circulating in the blood or lymph node system, can be beneficial in a variety of ways, such as reducing pain or discomfort, preventing metastasis, facilitating surgical intervention, and, more importantly, extending lifespan.

[0005] Various attempts have been made to support the immune system in fighting tumors. One early approach in the late 19th century involved general stimulation of the immune system, for example, by administering bacteria (live or dead) to induce a general immune response that would also be directed against tumors.

[0006] Recent approaches aim to help the immune system specifically recognize tumor-specific antigens (TSAs) (or tumor-associated antigens, TAAs) by administering them to the subject, usually in combination with adjuvants. However, a lack of robust immune responses to TAAs is often observed in cancer. One factor contributing to a weak response to TAAs is the induction of inhibitory pathways / signals that suppress the immune response (often referred to as "immune checkpoints"). While such inhibitory signals are important for maintaining self-tolerance and protecting tissues from damage when the immune system responds to pathogenic infections, they can also reduce the body's ability to mount beneficial responses against tumor growth.

[0007] A new era of treatment has emerged with immune checkpoint inhibitors or blockers (ICBs) targeting inhibitory T cell receptors, such as CTLA-4, PD-L1, and PD-1 (Marabelle, OncoImmunology 2016). This rapidly growing field was even awarded the 2018 Nobel Prize in Medicine. These immunotherapeutic agents have provided favorable clinical outcomes in several advanced cancers, including lung cancer (Reck, NEJM 2016), melanoma (Robert, NEJM 2011), genitourinary cancers (Motzer, NEJM 2018), and head and neck cancer (Ferris, NEJM 2016). However, while primary resistance rates in patients with non-small cell lung cancer (NSCLC) range from 35–44%, secondary resistance rates approach 100% (Reck, NEJM 2016).

[0008] Several types of tumors are considered immunologically "cold"—that is, tumors that are unlikely to elicit a strong immune response and typically do not respond well to cancer immunotherapies such as ICB therapy (see, for example, Bonaventura et al., "Cold Tumors: A Therapeutic Challenge for Immunotherapy," Frontiers in Immunology, vol. 10, pp. 168 (2019)). These tumors are notably characterized by the lack of infiltration of tumor-antigen-specific immune cells, such as tumor-infiltrating lymphocytes (TILs), within the tumor.

[0009] Therefore, there is a need to develop new approaches to induce or stimulate immune responses against tumors, particularly those that are immunologically cold and / or resistant to immunotherapy.

[0010] This specification makes reference to a number of documents, the contents of which are incorporated herein by reference in their entireties. Summary of the Invention

[0011] In various aspects and embodiments, the present disclosure provides items 1-78 below.

[0012] 1. A method for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy, comprising administering to the subject an effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound has the formula A-(B): n wherein A is a peptide compound of 30 residues or less, which contains an amino acid sequence having at least 60% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 13, X1X2X3X4X5GVX6AKAGVX7NX8FKSESY (SEQ ID NO: 1) (X9) n GVX 10 AKAGVX 11 NX 12 FKSESY (SEQ ID NO: 2) YKX 13 LRRX 14 APRWDX 15 PLRDPALRX 16 X 17 L (SEQ ID NO: 3) YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 L (SEQ ID NO: 4) IKLSGGVQAKAGVINMDKSESM (SEQ ID NO: 5) IKLSGGVQAKAGVINMFKSESY (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESYK (SEQ ID NO: 7) GVQAKAGVINMFKSESY (SEQ ID NO: 8) GVRAKAGVRNMFKSESY (SEQ ID NO: 9) GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 10) YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 11) YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 13) X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 18 , and X 19 are independently selected from any amino acid, X 16 , X 17 , X 20 , and X 21 is independently selected from Q, P, Y, I, and L; n is 0, 1, 2, 3, 4 or 5 When X9 occurs more than once, each X9 is independently selected from any amino acid; X 19 When present multiple times, each X is independently selected from any amino acid; Optionally, the peptide compound is cyclic; B is at least one therapeutic agent, and B is attached to A directly or via a linker.

[0013] 2. The method of clause 1, wherein the peptide compound comprises or consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 13.

[0014] 3. The method of clause 1 or 2, wherein the peptide compound comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 13, and further comprises 1 to 3 additional amino acids at its amino and / or carboxy termini.

[0015] 4. The method of clause 3, wherein the peptide compound comprises a cysteine ​​residue at its amino and / or carboxy terminus.

[0016] 5. The peptide compound comprises or consists of one of the following amino acid sequences: Z1X1X2X3X4X5GVX6AKAGVX7NX8FKSESYZ2 (SEQ ID NO: 34) Z1(X9) n GVX 10 AKAGVX 11 NX 12 FKSESYZ2 (SEQ ID NO: 35) Z1YkX 13 LRRX 14 APRWDX 15 PLRDPALRX 16 X 17 LZ2 (SEQ ID NO: 36) Z1YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 LZ2 (SEQ ID NO: 37) Z1IKLSGGVQAKAGVINMDKSESMZ2 (SEQ ID NO: 38) Z1IKLSGGVQAKAGVINMFKSESYZ2 (SEQ ID NO: 39) Z1IKLSGGVQAKAGVINMFKSESYKZ2 (SEQ ID NO: 40) Z1GVQAKAGVINMFKSESYZ2 (SEQ ID NO: 41) Z1GVRAKAGVRNMFKSESYZ2 (SEQ ID NO: 42) Z1GVRAKAGVRN(Nle)FKSESYZ2 (SEQ ID NO: 43) Z1YKSLRRKAPRWDAPLRDPALRQLLZ2 (SEQ ID NO: 44) Z1YKSLRRKAPRWDAYLRDPALRQLLZ2 (SEQ ID NO: 45) Z1YKSLRRKAPRWDAYLRDPALRPLL Z2 (SEQ ID NO: 46), X1~X 21 is as defined in clause 1, Z1 is a cysteine ​​residue or is absent, Z2 is a cysteine ​​residue or is absent, and at least one of Z1 and Z2 is present.

[0017] 6. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 2.

[0018] 7. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 4.

[0019] 8. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO:5.

[0020] 9. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO:6.

[0021] 10. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO:7.

[0022] 11. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO:8.

[0023] 12. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0024] 13. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0025] 14. The method of clause 13, wherein the peptide compound comprises or consists of the amino acid sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 47).

[0026] 15. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0027] 16. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0028] 17. The method of clause 1 or 5, wherein said peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0029] 18. The method of any one of clauses 1 to 17, wherein the peptide compound comprises at least one modifying group at its amino terminus and / or carboxy terminus.

[0030] 19. The method of clause 17, wherein the at least one modifying group is acetyl or succinyl.

[0031] 20. The method of clause 1, wherein the peptide compound is represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 48. Acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14) Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15) Acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16) Acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17) Acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18) Acetyl-GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 48).

[0032] 21. The method of any one of clauses 1 to 20, wherein B is attached to A at a free amine of the peptide compound, at the N-terminal position of the peptide compound, at a free -SH of the peptide compound, and / or at a free carboxyl of the peptide compound.

[0033] 22. The method of any one of clauses 1 to 21, wherein B is attached to A via a linker.

[0034] 23. The conjugate is represented by formula (LIII) or (LIV): GVRAK(J 1 )AGVRN(Nle)FK(J 2 ) SESY(LIII) (SEQ ID NO: 22), Acetyl-GVRAK(J 1 )AGVRN(Nle)FK(J 2 ) SESY(LIV) (SEQ ID NO: 23), During the ceremony, J. 1 and J 2 23. The method of any one of clauses 1 to 22, wherein each is independently a therapeutic agent attached to a lysine (K) residue.

[0035] 24. The method of any one of clauses 1 to 23, wherein said therapeutic agent is an anti-tumor agent, such as a radionuclide or a chemotherapeutic agent.

[0036] 25. The method of clause 24, wherein the chemotherapeutic agent is a taxane.

[0037] 26. The method of clause 25, wherein the chemotherapeutic agent is docetaxel.

[0038] 27. The method of any one of clauses 1-26, further comprising treating said subject with immunotherapy.

[0039] 28. The method of any one of clauses 1 to 27, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0040] 29. The method of clause 28, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0041] 30. The method of clause 28 or 29, wherein said ICI is a blocking antibody.

[0042] 31. The method of clause 28 or 29, wherein said ICI is a PD-L1 inhibitor.

[0043] 32. The method of any one of clauses 1 to 31, wherein said cancer is an immunologically cold cancer.

[0044] 33. The method of any one of clauses 1 to 32, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0045] 34. Use of a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

[0046] 35. Use of a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

[0047] 36. The use according to clause 34 or 35, wherein the conjugate compound, its pharmaceutically acceptable salt, or medicament is for use in combination with immunotherapy.

[0048] 37. The use according to any one of clauses 34 to 36, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0049] 38. The use according to clause 37, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0050] 39. The use according to clause 37 or 38, wherein said ICI is a blocking antibody.

[0051] 40. The use according to clause 38 or 39, wherein said ICI is a PD-L1 inhibitor.

[0052] 41. The use according to any one of clauses 34 to 40, wherein the cancer is an immunologically cold cancer.

[0053] 42. The use according to any one of clauses 34 to 41, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0054] 43. A conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof for use in (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

[0055] 44. A conjugate compound or a pharmaceutically acceptable salt thereof for use as described in clause 43, wherein said peptide compound is for use in combination with immunotherapy.

[0056] 45. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to clause 43 or 44, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0057] 46. ​​The conjugate compound or a pharmaceutically acceptable salt thereof for use according to clause 45, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0058] 47. A conjugate compound or a pharmaceutically acceptable salt thereof for use according to clause 45 or 46, wherein said ICI is a blocking antibody.

[0059] 48. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to clause 46 or 47, wherein said ICI is a PD-L1 inhibitor.

[0060] 49. A conjugate compound or a pharmaceutically acceptable salt thereof for use according to any one of clauses 43 to 48, wherein said cancer is an immunologically cold cancer.

[0061] 50. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to any one of clauses 43 to 49, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0062] 51. A method for treating a sortilin-expressing cancer in a subject, the method comprising administering to said subject a therapeutically effective amount of a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof in combination with immunotherapy.

[0063] 52. The method of clause 51, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0064] 53. The method according to clause 52, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0065] 54. The method of clause 52 or 53, wherein said ICI is a blocking antibody.

[0066] 55. The method according to clause 53 or 54, wherein said ICI is a PD-L1 inhibitor.

[0067] 56. The method of any one of clauses 51 to 55, wherein said cancer is an immunologically cold cancer.

[0068] 57. The method of any one of clauses 51 to 56, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0069] 58. The method of any one of clauses 51 to 57, wherein said conjugate compound or pharmaceutically acceptable salt thereof and said immunotherapy are present in different compositions.

[0070] 59. The method of any one of clauses 51 to 57, wherein said conjugate compound or pharmaceutically acceptable salt thereof and said immunotherapy are present in the same composition.

[0071] 60. Use of a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof in combination with immunotherapy for the treatment of sortilin-expressing cancer.

[0072] 61. Use of a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof in combination with immunotherapy for the manufacture of a medicament for the treatment of sortilin-expressing cancer.

[0073] 62. The use according to clause 60 or 61, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0074] 63. The use according to clause 62, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0075] 64. The use according to clause 62 or 63, wherein said ICI is a blocking antibody.

[0076] 65. The use according to clause 63 or 64, wherein said ICI is a PD-L1 inhibitor.

[0077] 66. The use according to any one of clauses 60 to 65, wherein the cancer is an immunologically cold cancer.

[0078] 67. The use according to any one of clauses 61 to 66, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0079] 68. The use according to any one of clauses 61 to 67, wherein said conjugate compound or a pharmaceutically acceptable salt thereof and said immunotherapy are present in different compositions.

[0080] 69. The use according to any one of clauses 61 to 67, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are present in the same composition.

[0081] 70. A combination therapy for use in the treatment of sortilin-expressing cancer, comprising a conjugate compound as defined in any one of clauses 1 to 26 or a pharmaceutically acceptable salt thereof, and immunotherapy.

[0082] 71. The combination therapy for use as described in clause 70, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0083] 72. Combination therapy for use according to clause 71, wherein said ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0084] 73. Combination therapy for use according to clause 71 or 72, wherein said ICI is a blocking antibody.

[0085] 74. The combination therapy for use according to clause 72 or 73, wherein said ICI is a PD-L1 inhibitor.

[0086] 75. Combination therapy for use according to any one of clauses 71 to 74, wherein said cancer is an immunologically cold cancer.

[0087] 76. The combination therapy for use according to any one of clauses 70 to 75, wherein said cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0088] 77. A combination therapy for use according to any one of clauses 70 to 76, wherein said conjugate compound or a pharmaceutically acceptable salt thereof and said immunotherapy are present in different compositions.

[0089] 78. A combination therapy for use according to any one of clauses 70 to 76, wherein said conjugate compound or a pharmaceutically acceptable salt thereof and said immunotherapy are present in the same composition. Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of specific embodiments of the invention, given by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0090] [Figure 1]Figure 1A shows the sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment halted 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by three cycles off treatment. Tumor growth was monitored for the indicated days as described in the Examples. Data are presented as mean ± SEM (3 mice / group). Representative tumor sections were stained for H&E (Figure 1B) or SORT1, Ki67, STING, and CD45 (Figure 1C), as described in Example 1. [Figure 1B] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment halted 4 days after (i) three cycles or, in the case of TH1902, after (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by three cycles off treatment. Representative tumor sections were stained for H&E (Figure 1B) or for SORT1, Ki67, STING, and CD45 (Figure 1C), as described in Example 1. [Figure 1C] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5].Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment halted 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by a three-cycle break. Representative tumor sections were stained for H&E (Figure 1B) or for SORT1, Ki67, STING, and CD45 (Figure 1C), as described in Example 1. [Figure 1D] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model is shown. In vivo MDA-MB-231 TNBC xenograft models were generated in immunodeficient nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment stopped 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 (iii) six cycles followed by a three-cycle break. Vascular mimicry was assessed by monitoring CD31- / PAS+ staining, while normal vasculature was assessed by CD31+ / PAS+ staining. [Figure 1E] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model is shown. In vivo MDA-MB-231 TNBC xenograft models were generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment stopped 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by three rest cycles. Mouse weights were monitored as described in Example 1. Mouse weights are expressed as a percentage of the initial weight at the start of treatment (day 1). Data are presented as mean ± SEM (3 mice / group).[Figure 1F] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment halted 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by a three-cycle break. Differential effects of TH1902 and docetaxel on p21 and p53 expression in MDA-MB-231 cells were observed. MDA-MB-231 cells were treated with vehicle (DMSO), 50 nM docetaxel, or TH1902 for 5 minutes, followed by incubation in fresh complete medium for 96 hours. Cell lysates were collected as described in Example 1, and representative immunoblots were performed using anti-p21, anti-p53, and anti-GAPDH antibodies (Figure 1F). Protein expression was quantified using densitometry (Figure 1G). [Figure 1G] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model was demonstrated. In vivo MDA-MB-231 TNBC xenograft models were generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment stopped 4 days after (i) three cycles or, in the case of TH1902, (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by three cycles off treatment. Differential effects of TH1902 and docetaxel on p21 and p53 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with vehicle (DMSO) or 50 nM docetaxel or TH1902 for 5 minutes, followed by incubation in fresh complete medium for 96 hours.Cell lysates were collected as described in Example 1, and representative immunoblots were performed using anti-p21, anti-p53, and anti-GAPDH antibodies (Figure 1F). Protein expression was quantified using densitometry (Figure 1F). [Figure 1H] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model was demonstrated. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment halted 4 days after (i) three cycles or, in the case of TH1902, after (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by a three-cycle break. PD-L1 quantification of MDA-MB-231 / Luc tumors measured in vivo using QuPath software. % of PD-L1-positive areas. [Figure 1I] Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model. An in vivo MDA-MB-231 TNBC xenograft model was generated in immunocompromised nude mice as previously described [5]. Mice were treated intravenously with either docetaxel at the MTD of 15 mg / kg / week or TH1902 at 35 mg / kg / week, with treatment stopped 4 days after (i) three cycles or, in the case of TH1902, after (ii) six cycles. An additional group was treated with TH1902 for (iii) six cycles followed by a three-cycle break. PD-L1 quantification of MDA-MB-231 / Luc tumors measured in vivo using QuPath software. % of PD-L1-positive cells. [Figure 2]Figure 2A shows SORT1 expression in melanoma tissues and cell line models. Tissue microarrays of clinically annotated stage II-IV melanoma and healthy tissues were evaluated for SORT1 expression by immunohistochemistry. Figure 2B shows SORT1 expression in melanoma tissues and cell line models. IHS scoring was performed as described in Example 1 (normal n=2, stage II n=2, stage III n=3, stage IV n=5). Figure 2C shows SORT1 expression in melanoma tissues and cell line models. Cell lysates (20 μg) from different cancer cell lines were evaluated for SORT1 expression levels by Western blot analysis. B16-F10: mouse B16-F10 melanoma cells, SKMEL: human SK-MEL-28 melanoma cells, A375: human A375 melanoma cells, and MDA: human TNBC-derived MDA-MB-231 cells. [Figure 3][Figure 3A] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Cell proliferation in response to docetaxel or TH1902 was evaluated in SK-MEL-28 (Figure 3A) and B16-F10 (Figure 3B) melanoma cells as described in Example 1, and IC50 values ​​for each test substance were extracted (Figure 3C). [Figure 3B] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Cell proliferation in response to docetaxel or TH1902 was evaluated in SK-MEL-28 (Figure 3A) and B16-F10 (Figure 3B) melanoma cells as described in Example 1, and IC50 values ​​for each test substance were extracted (Figure 3C). [Figure 3C] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Cell proliferation in response to docetaxel or TH1902 was evaluated in SK-MEL-28 (Figure 3A) and B16-F10 (Figure 3B) melanoma cells as described in Example 1, and IC50 values ​​for each test substance were extracted (Figure 3C). [Figure 3D] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Cell apoptosis was evaluated in B16-F10 melanoma cells after treatment with docetaxel (black bars) and TH1902 (gray bars) at the indicated concentrations. [Figure 3E] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Fluorescence microscopy was used to evaluate cellular senescence in B16-F10 melanoma cells after treatment with docetaxel, TH1902, or etoposide (as a positive control for cellular senescence induction), and representative images are shown. [Figure 3F] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells. Senescence-associated β-galactosidase activity was quantified as described in Example 1 (n=3). [Figure 3G] TH1902 exhibits antiproliferative and apoptotic activity in vitro and induces senescence in SORT1-positive melanoma cells.Changes in cell morphology were assessed using crystal violet staining of cells treated with vehicle (DMSO), 100 nM docetaxel, or 50 nM TH1902 (equivalent docetaxel content). [Figure 4][Figure 4A] Leukocyte infiltration within B16-F10 tumors treated with vehicle, docetaxel, or TH1902. Tumor growth in syngeneic mice treated with vehicle, 15 mg / kg / week docetaxel (MTD), or 35 mg / kg / week TH1902 (equivalent docetaxel content). Data are shown as mean ± SEM (9 mice / group for vehicle and docetaxel, 10 mice / group for TH1902). [Figure 4B] Leukocyte infiltration within B16-F10 tumors treated with vehicle, docetaxel, or TH1902. B16-F10 melanoma tumors were excised and photographed. [Figure 4C] Leukocyte infiltration within B16-F10 tumors treated with vehicle, docetaxel, or TH1902. The excised tumors were fixed in formalin and processed for immunohistochemical analysis. The top layer of the image was stained with hematoxylin and eosin, and the bottom layer was immunohistochemically evaluated using a monoclonal antibody against CD45 (a panclonal immune cell). Representative images of the whole and enlarged tumors for both stainings are shown (black scale bar = 2 mm, white scale bar = 100 μm). [Figure 4D] Leukocyte infiltration within B16-F10 tumors treated with vehicle, docetaxel, or TH1902 is shown. The amount of CD45 stained area was compared for the three mouse groups using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. The area positively stained for leukocytes was significantly greater in TH1902-treated animals than in vehicle- or docetaxel-treated animals. Data are shown as mean ± SEM (*p<0.05, **p<0.01, n=4 tumors analyzed per group). [Figure 4E] Leukocyte infiltration within B16-F10 tumors treated with vehicle, docetaxel, or TH1902. Mouse body weights in syngeneic mice treated with vehicle, 15 mg / kg / week docetaxel (MTD), or 35 mg / kg / week TH1902 (equivalent docetaxel content). Mouse body weights are expressed as a percentage of the initial body weight at the start of treatment (day 1). Data are shown as mean ± SEM (9 mice / group for vehicle and docetaxel, 10 mice / group for TH1902). [Figure 5]Figure 5A shows the effect of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages in syngeneic tumors. Each row of photographic microscopy images represents cells from B16-F10 tumors of animals treated with vehicle, docetaxel, or TH1902 (same samples as in Figures 4A–E). Each column contains representative IHC images of immune cell markers CD3 (T cells), CD8 (cytotoxic T cells), CD4 (helper T cells), FoxP3 (regulatory T cells), and CD161c (NK cells) performed on paraffin sections of the primary tumor (white scale bar = 100 μm). Figure 5B shows the effect of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages in syngeneic tumors. Quantification of IHC staining from Figure 5A. Expression was compared using one-way ANOVA and Tukey's multiple comparison test. Data are shown as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001; n=4 tumors analyzed per group). Figures 5C-D show quantification of immune cell infiltration in B16-F10 xenograft tumors treated with docetaxel and TH1902. [Figure 5C] Effect of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages in syngeneic tumors. [Figure 5D] Effect of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages in syngeneic tumors. [Figure 5E] Effect of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages in syngeneic tumors. Figure 1 shows quantification of immune cell infiltration in B16-F10 xenograft tumors treated with docetaxel and TH1902. Tumor-associated macrophages (TAMs) include M1 (CD68+) and M2 (CD206+) macrophages. [Figure 6]Figure 6A shows the effect of docetaxel and TH1902 on the expression of tumor markers for immune-stimulated apoptosis. Each row of photographic microscopy images represents cells from B16-F10 tumors of animals treated with vehicle, docetaxel, or TH1902 (same samples as in Figure 4). Each column contains representative IHC images of markers involved in immune-stimulated apoptosis, including cleaved caspase 3, perforin, and granzyme B (white scale bar = 100 μm). Figure 6B shows the effect of docetaxel and TH1902 on the expression of tumor markers for immune-stimulated apoptosis. Quantification of IHC staining from Figure 6A. Expression was compared using one-way ANOVA and Tukey's multiple comparison test. Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, n = 4 tumors analyzed per group). [Figure 7]Figure 7A shows the effect of TH1902 in combination with checkpoint inhibitors on tumor growth and mouse survival. B16-F10 cells were subcutaneously implanted into immunocompetent C57BL / 6 mice. The effect of docetaxel, TH1902, and anti-PD-L1, alone or in combination, on B16-F10 tumor growth. Mice were treated intravenously once weekly with vehicle, docetaxel (7.5 mg / kg), or TH1902 (docetaxel-equivalent dose 17.5 mg / kg), or intraperitoneally every other week with anti-PD-L1 (9 mg / kg) and control isotype (9 mg / kg), alone or in combination (docetaxel / anti-PD-L1 or TH1902 / anti-PD-L1), for two cycles. Data are presented as mean ± SEM (n = 8 mice per group). Figure 7B shows the effect of TH1902 in combination with checkpoint inhibitors on tumor growth and mouse survival. B16-F10 cells were subcutaneously implanted into immunocompetent C57BL / 6 mice. The effect of increasing doses of TH1902 and anti-PD-L1, alone or in combination, on survival (Figure 7B) and tumor growth (Figure 7C) in B16-F10 tumor-bearing mice. Mice were treated intravenously once weekly with vehicle or TH1902 (4.37, 8.75, 17.5 mg / kg), or every two weeks with anti-PD-L1 (9 mg / kg) and control isotype (9 mg / kg), alone or in combination, in successive treatment cycles until one of the defined endpoints was reached as described in Example 1. Kaplan-Meier curves were plotted to estimate mouse survival, showing the probability of survival as a percentage (top panel), while tumor growth curves were plotted until one individual within a particular group reached a tumor size endpoint (tumor >2,000 mm3, bottom panel). Data are presented as mean ± SEM (n = 6 mice / group). [Figure 7C] The effect of TH1902 in combination with checkpoint inhibitors on tumor growth and mouse survival is shown. B16-F10 cells were subcutaneously implanted into immunocompetent C57BL / 6 mice.The effect of increasing doses of TH1902 and anti-PD-L1, alone or in combination, on survival (Figure 7B) and tumor growth (Figure 7C) in mice bearing B16-F10 tumors. Mice were treated intravenously once weekly with vehicle or TH1902 (4.37, 8.75, or 17.5 mg / kg), or every two weeks with anti-PD-L1 (9 mg / kg) and control isotype (9 mg / kg), alone or in combination, in successive treatment cycles until one of the defined endpoints was reached as described in Example 1. Kaplan-Meier curves were plotted to estimate mouse survival, showing the probability of survival as a percentage (top panel), while tumor growth curves were plotted until one individual within a particular group reached a tumor size endpoint (tumor >2,000 mm3, bottom panel). Data are presented as mean ± SEM (n = 6 mice / group). [Figure 7D] The effect of TH1902 in combination with checkpoint inhibitors on tumor growth and mouse survival. B16-F10 cells were subcutaneously implanted into immunocompetent C57BL / 6 mice. Mouse body weights are expressed as a percentage of the initial body weight at the start of treatment (day 1). Data are presented as mean ± SEM (n = 8 mice / group). [Figure 8] Figure 1 shows the effect of various checkpoint inhibitors on B16-F10 melanoma tumor growth (corresponding to Figure 1A in Ueha et al., Cancer Immunol Res (2015) 3(6):631-640). Mice bearing B16-F10 melanoma tumors were intraperitoneally injected with anti-CD4 mAb (200 mg / mouse) on days 5 and 9, and with anti-immune checkpoint mAbs (anti-PD1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-OX40, anti-LAG-3, anti-TIM-3, anti-BTLA, or anti-GITR) on days 4, 8, 14, and 18 after tumor inoculation. [Figure 9]Figure 9A shows that TH1902 induces the cell surface expression of downstream effectors of the STING pathway and PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours. Cell lysates were collected as described in Example 1, immunoblotted using anti-STING and anti-GAPDH antibodies (Figure 9A), and protein expression was quantified using densitometry (Figure 9B). Figure 9B shows that TH1902 induces the cell surface expression of downstream effectors of the STING pathway and PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours. Cell lysates were collected as described in Example 1, immunoblotted using anti-STING and anti-GAPDH antibodies (Figure 9A), and protein expression was quantified using densitometry (Figure 9B). [Figure 9C] TH1902 induces the cell surface expression of downstream effectors of the STING pathway and PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours. Lysates were further processed for the indicated expression of STING downstream effectors in 100 nM docetaxel or TH1902, followed by densitometric quantification (Figure 9D). [Figure 9D] TH1902 induces the cell surface expression of downstream effectors of the STING pathway and PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours.Lysates were further treated with 100 nM docetaxel or TH1902 for the indicated expression of STING downstream effectors, and densitometric quantification was performed (Figure 9D). [Figure 9E] This figure shows that TH1902 induces the cell surface expression of downstream effectors of the STING pathway and PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours. Total RNA was extracted, and gene expression of IL-6 and TNFα was assessed using RT-qPCR in cells treated with 100 nM docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 24 hours. Figure 9F shows that TH1902 induces downstream effectors of the STING pathway and cell surface expression of PD-L1 and MHC-I in B16-F10 melanoma cells. Cells were treated with increasing concentrations of docetaxel or TH1902 for 5 minutes, then incubated in fresh complete medium for 96 hours. Immunophenotypic analysis of MHC-I and PD-L1 cell surface expression was performed by flow cytometry as described in Example 1. [Figure 10] Figure 10A shows that TNFα, but not IL-6, induces PD-L1 and MHC-I gene expression in B16-F10 melanoma cells. Cells were treated with the indicated concentrations of TNFα or IL-6 for 96 hours. PD-L1 and MHC-I expression levels were estimated by flow cytometry as described in the Methods section. PD-L1 and MHC-I cell surface expression upon TNFα treatment (Figure 10A) or IL-6 treatment (Figure 10B). Figure 10B shows that TNFα, but not IL-6, induces PD-L1 and MHC-I gene expression in B16-F10 melanoma cells. Cells were treated with the indicated concentrations of TNFα or IL-6 for 96 hours. PD-L1 and MHC-I expression levels were estimated by flow cytometry as described in the Methods section. PD-L1 and MHC-I cell surface expression upon TNFα treatment (Figure 10A) or IL-6 treatment (Figure 10B). [Figure 11][Figure 11A] Depicts the effect of treatment with checkpoint inhibitors anti-PD1 (Figure 11A) and anti-PD-L1 (Figure 11B) in a LL / 2 (Lewis lung) lung cancer model (https: / / drugdevelopment.labcorp.com / industry-solutions / oncology / preclinical / tumor-spotlights / ll-2-an-immunosuppressive-murine-tumor-model.html, from Figure 5). [Figure 11B] Depicts the effect of treatment with checkpoint inhibitors anti-PD1 (Figure 11A) and anti-PD-L1 (Figure 11B) in a LL / 2 (Lewis lung) lung cancer model (https: / / drugdevelopment.labcorp.com / industry-solutions / oncology / preclinical / tumor-spotlights / ll-2-an-immunosuppressive-murine-tumor-model.html, from Figure 5). [Figure 11C] The effects of TH1902 and docetaxel on LL / 2 lung cancer xenografts. LL / 2 cells were subcutaneously implanted into the dorsum of C57BL / 6 mice, and treatment was performed 3 days later. Tumor volume was measured after weekly administration of vehicle, docetaxel (15 mg / kg), or TH1902 (35 mg / kg). Data are presented as mean ± SEM (n = 7 mice / group). Exponential curve analysis (hypersum-of-squares F test) indicates that all curves are significantly different from each other. [Figure 12] The amino acid sequence of human sortilin-1 (SORT1, UniProtKB accession No. Q99523) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0091] In the context of describing the present invention (particularly in the context of the claims which follow), the use of the terms "a," "an," and "the" and similar referents should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0092] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0093] Recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually referred to herein. Every subset of values ​​within a range is also incorporated herein as if it were individually listed herein.

[0094] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0095] Any examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to facilitate understanding of the invention and do not limit the scope of the invention unless otherwise required.

[0096] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0097] As used herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation for error of the device or method being used to determine the value, or encompasses a value near the recited value, e.g., within 10% of the recited value (or range of values).

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0099] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0100] The present invention includes all combinations and subcombinations of the embodiments and features disclosed herein.

[0101] In the studies described herein, the inventors showed that in a mouse model of immunologically cold tumors, a sortilin-targeting peptide conjugated to a chemotherapeutic drug (docetaxel) can induce infiltration of antitumor immune cells into tumors, suppress tumor growth, and enhance the antitumor response of immune checkpoint inhibitors.

[0102]

[0010] Accordingly, in a first aspect, the present disclosure provides a method for (i) enhancing an anti-tumor immune response in a subject suffering from cancer and / or (ii) treating a subject suffering from cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy), the method comprising administering to the subject an effective amount of a conjugate compound as defined herein, or a pharmaceutically acceptable salt thereof. The present disclosure also provides the use of a conjugate compound as defined herein, or a pharmaceutically acceptable salt thereof, for (i) enhancing an anti-tumor immune response in a subject suffering from cancer and / or (ii) treating a subject suffering from cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). The present disclosure also provides the use of a conjugate compound as defined herein, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for (i) enhancing an anti-tumor immune response in a subject suffering from cancer and / or (ii) treating a subject suffering from cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). The present disclosure also provides a binding compound as defined herein, or a pharmaceutically acceptable salt thereof, for use in (i) enhancing an anti-tumor immune response in a subject suffering from cancer, and / or (ii) treating a subject suffering from cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0103] In one embodiment, the method or use is for enhancing an anti-tumor immune response in a subject suffering from cancer. In another embodiment, the method or use is for treating a subject suffering from a cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). In another embodiment, the method or use is for enhancing an anti-tumor immune response in a subject suffering from a cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0104] Conjugates suitable for the methods and uses disclosed herein are those that include a therapeutic agent, such as an anti-tumor agent (e.g., a chemotherapeutic agent), conjugated to an agent, such as a peptide capable of binding to Sortilin, which is taken up by tumor cells, thereby delivering the anti-tumor agent to the tumor cells. In some embodiments, the conjugate (or conjugate compound) includes an anti-Sortilin antibody or antigen-binding fragment thereof conjugated to an anti-tumor agent (i.e., an antibody-drug conjugate, ADC). Examples of anti-Sortilin antibodies and antigen-binding fragments thereof include anti-Sortilin monoclonal antibody clone 2D8-E3 (Ghaemimanesh F. et al., Monoclon Antib Immunodiagn Immunother 2015;34(6):390-5), and antibodies and antigen-binding fragments thereof disclosed in PCT Publication Nos. WO2016 / 164637, WO2017 / 009327, WO2019 / 016247, WO2020 / 014617, WO2020 / 252066, WO2021 / 116290, WO2021 / 263279, WO2022 / 261648, and WO2023 / 122766, the contents of which are incorporated herein by reference.

[0105] Peptide compounds that bind to sortilin are disclosed in PCT Publication Nos. WO2017 / 088058, WO2018 / 213928, WO2020 / 037434, WO2021 / 078833, WO2021 / 028404, and WO2023 / 165476, the contents of which are incorporated herein by reference.

[0106] In one embodiment, the conjugate (or conjugate compound) is an antitumor agent-peptide compound conjugate described in PCT Publication Nos. WO2017 / 088058, WO2018 / 213928, and WO2020 / 037434.

[0107] As used herein, the terms "sortilin" or "sortilin receptor" refer to a neuronal type 1 membrane glycoprotein encoded by the SORT1 gene and belonging to the vaccinia protein sorting 10 (Vps10) family of receptors. Sortilin (also known as neurotensin receptor 3; UniProtKB accession number Q99523) is expressed or overexpressed in several cancers, including ovarian, breast, colon, and prostate cancers. The encoded precursor protein (residues 34-831, with residues 1-33 corresponding to the signal peptide) is proteolytically processed by furin (or other homologous proteases) after amino acid 77 to generate the mature receptor (residues 78-831) with a molecular mass of approximately 100-110 kDa. The amino acid residues of sortilin shown herein correspond to the positions in the full-length form (i.e., UniProtKB accession number Q99523, Figure 12).

[0108] In certain embodiments, the conjugate compound has the formula A-(B): n and wherein A is a peptide compound comprising an amino acid sequence having at least 60% sequence identity with any of the sequences of formulae (I)-(XIII), X1X2X3X4X5GVX6AKAGVX7NX8FKSESY(I) (SEQ ID NO: 1) (X9) n GVX 10 AKAGVX 11 NX 12 FKSESY(II) (SEQ ID NO: 2) YkX 13 LRRX 14 APRWDX 15 PLRDPALRX 16 X 17 L(III) (SEQ ID NO: 3) YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 L(IV) (SEQ ID NO: 4) IKLSGGVQAKAGVINMDKSESM(V) (SEQ ID NO: 5) IKLSGGVQAKAGVINMFKSESY (VI) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESYK(VII) (SEQ ID NO: 7) GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 8) GVRAKAGVRNMFKSESY (IX) (SEQ ID NO: 9) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 10) YKSLRRKAPRWDAPLRDPALRQLL(XI) (SEQ ID NO: 11) YKSLRRKAPRWDAYLRDPALRQLL (XII) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRPLL (XIII) (SEQ ID NO: 13) During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 18 and X 19 are independently selected from any amino acid, and X 16 , X 17 , X 20 and X 21 is independently selected from Q, P, Y, I, and L, n is an integer from 1 to 10, e.g., 1, 2, 3, 4, or 5, and when X9 occurs multiple times, each X9 is independently selected from any amino acid; 19 When occurs multiple times, each X9 is independently selected from any amino acid, and optionally the peptidic compound is cyclic, and the peptidic compound binds to sortilin.

[0109] B is at least one therapeutic agent, such as a tumor therapeutic agent, and B is bonded to A directly or via a linker, or a pharmaceutically acceptable salt thereof, optionally at a free amine of the peptide compound, an N-terminal position of the peptide compound, a free —SH of the peptide compound, or a free carboxyl of the peptide compound.

[0110] The term "amino acid" refers to common naturally occurring (genetically encoded) or synthetic amino acids and their common derivatives known to those of skill in the art. When applied to amino acids, "standard" or "proteinogenic" refers to the 20 genetically encoded amino acids in their natural configuration. Similarly, when applied to amino acids, "nonstandard," "non-natural," or "unusual" refers to the wide selection of non-natural, rare, or synthetic amino acids, as described by Hunt, S., in Chemistry and Biochemistry of the Amino Acids, Barrett, GC, ed., Chapman and Hall: New York, 1985. Some examples of non-standard amino acids include non-alpha amino acids and D-amino acids. In one embodiment, the peptide compound contains only natural amino acids. In another embodiment, the peptide compound contains one or more non-natural or synthetic amino acids, such as D-amino acids.

[0111] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences can be accomplished using a mathematical algorithm. To obtain amino acid sequences homologous to the protein molecules of the present disclosure, BLAST protein searches can be performed, for example, using the XBLAST program parameters set to score = -50 and word length = 3. Gapped BLAST can be used to obtain gapped alignments for comparison purposes. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0112] The term "pharmaceutically acceptable" means compatible with the treatment of a subject, such as an animal or a human. Also described herein are pharmaceutically acceptable salts of the conjugate compounds described herein. The term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt that is compatible with or adaptable for the treatment of a subject, such as an animal or a human. As used herein, the term "pharmaceutically acceptable acid addition salt" refers to a non-toxic organic or inorganic salt of any compound in the present disclosure or its intermediates. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, as well as metal salts such as sodium monohydrogen phosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include mono-, di-, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Mono- or di-acid salts may be formed, and such salts may exist in hydrated, solvated, or nearly anhydrous forms. Generally, acid addition salts of the disclosed compounds are more soluble in water and various hydrophilic organic solvents and generally have higher melting points compared to the free base form. The selection of an appropriate salt will be known to those skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used in the isolation of the disclosed compounds for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts. As used herein, the phrase "pharmaceutically acceptable acid addition salt" refers to a non-toxic organic or inorganic base addition salt of any disclosed acid compound or its intermediate. The disclosed acid compounds can form base addition salts, for example, when COH is a functional group. Exemplary inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, picoline, or ammonia. The selection of an appropriate salt will be known to those skilled in the art.Other non-pharmaceutically acceptable base addition salts may be used in the isolation or conjugation of compounds of the present disclosure for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0113] In certain embodiments, the peptide compound comprises or consists of a sequence of any of Formulas (I)-(XIII). In certain embodiments, the peptide compound comprises or consists of an amino acid sequence of any of SEQ ID NOs: 1-13. In embodiments, the peptide compound comprises no more than 50, 45, 40, 35, 30, 25, or 20 amino acids.

[0114] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (I) or SEQ ID NO:1, and wherein the peptidic compound binds to Sortilin.

[0115] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (II) or SEQ ID NO:2, and wherein the peptidic compound binds to Sortilin.

[0116] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (III) or SEQ ID NO:3, and wherein the peptidic compound binds to Sortilin.

[0117] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a peptidic compound of Formula (IV) or SEQ ID NO:4, and wherein the peptidic compound binds to Sortilin.

[0118] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a peptidic compound of Formula (V) or SEQ ID NO:5, and wherein the peptidic compound binds to Sortilin.

[0119] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (VI) or SEQ ID NO:6, and wherein the peptidic compound binds to Sortilin.

[0120] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (VII) or SEQ ID NO:7, and wherein the peptidic compound binds to Sortilin.

[0121] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (VIII) or SEQ ID NO:8, and wherein the peptidic compound binds to Sortilin.

[0122] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (IX) or SEQ ID NO:9, and wherein the peptidic compound binds to Sortilin.

[0123] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (X) or SEQ ID NO: 10, and wherein the peptidic compound binds to Sortilin.

[0124] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (XI) or SEQ ID NO:11, and wherein the peptidic compound binds to Sortilin.

[0125] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (XII) or SEQ ID NO: 12, and wherein the peptidic compound binds to Sortilin.

[0126] In embodiments, the peptidic compound comprises or consists of an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a peptidic compound of Formula (XIII) or SEQ ID NO: 13, and wherein the peptidic compound binds to Sortilin.

[0127] In one embodiment, the peptide compound contains no more than 30, 25, or 20 residues and comprises the sequence GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 10). In another embodiment, the peptide compound contains no more than 30, 25, or 20 residues and comprises the sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 31).

[0128] In some embodiments, at least one modifying group is attached to the N-terminus and / or C-terminus of the peptide compound. In some embodiments, the peptide compound comprises a modifying group at the N-terminus. In some embodiments, the peptide compound comprises a modifying group at the C-terminus. Such modifying groups can serve to protect the peptide compound from modification or degradation (e.g., degradation by proteases). In one embodiment, the amino-terminal modifying group is C1-C 16 or C3-C 16 The amino terminal modifying group is an acyl group (linear or branched, saturated or unsaturated), and in further embodiments, a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched). In another embodiment, the amino terminal modifying group is an acetyl group (CH3-CO-, Ac) or a succinyl group (CO-CH2-CH2-CO-). The carboxyl terminal modifying group can be, for example, a hydroxylamine group (NHOH) attached to the carboxyl group (-C(=O)-NHOH) or an amine attached to the carboxyl group (-C(=O)-NRR'), where the amine is a primary, secondary, or tertiary amine, preferably an alkylamine having 1 to 10 carbon atoms, such as methylamine, isobutylamine, isovaleramine, or cyclohexylamine, or an aromatic amine or arylalkylamine, such as aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine, with the preferred amine being -NH2.

[0129] In one embodiment, a sucinyl group is attached to the peptide compound, for example, the peptide compound has the sequence sucinyl-IKLSGGVQAKAGVINMFKSESY, corresponding to SEQ ID NO: 6, and is attached to the N-terminus.

[0130] In one embodiment, an acetyl group is attached to the peptide compound. For example, the peptide compound has the sequence acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14). For example, the peptide compound has the sequence acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15). For example, the peptide compound has the sequence acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16). For example, the peptide compound has the sequence acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17). For example, the peptide compound has the sequence acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18).

[0131] In one embodiment, the peptide compound can be modified at the C-terminus and / or N-terminus by the addition of amino acid residues (e.g., 1-5 or 1-3) to obtain or increase preferential binding sites at the peptide termini. For example, the amino acid can be cysteine. For example, the amino acid can be lysine. For example, the amino acid can be cysteine ​​added to the N-terminus and / or C-terminus of the peptide. In one embodiment, the peptide compound is modified by adding a cysteine ​​at the C-terminus.

[0132] Thus, in embodiments, the peptide compound comprises or consists of any of the following amino acid sequences: Z1X1X2X3X4X5GVX6AKAGVX7NX8FKSESYZ2 (SEQ ID NO: 34) Z1(X9) n GVX 10 AKAGVX 11 NX 12 FKSESYZ2 (SEQ ID NO: 35) Z1YkX 13 LRRX 14 APRWDX 15 PLRDPALRX 16 X 17 LZ2 (SEQ ID NO: 36) Z1YkX 18 LRR(X 19 ) N PLRDPALRX20 X 21 LZ2 (SEQ ID NO: 37) Z1IKLSGGVQAKAGVINMDKSESMZ2 (SEQ ID NO: 38) Z1IKLSGGVQAKAGVINMFKSESYZ2 (SEQ ID NO: 39) Z1IKLSGGVQAKAGVINMFKSESYKZ2 (SEQ ID NO: 40) Z1GVQAKAGVINMFKSESYZ2 (SEQ ID NO: 41) Z1GVRAKAGVRNMFKSESYZ2 (SEQ ID NO: 42) Z1GVRAKAGVRN(Nle)FKSESYZ2 (SEQ ID NO: 43) Z1YKSLRRKAPRWDAPLRDPALRQLLZ2 (SEQ ID NO: 44) Z1YKSLRRKAPRWDAYLRDPALRQLLZ2 (SEQ ID NO: 45) Z1YKSLRRKAPRWDAYLRDPALRPLLZ2 (SEQ ID NO: 46) X1~X 21 are as defined above, Z1 is a cysteine ​​residue or is absent, and Z2 is a cysteine ​​residue or is absent. In some embodiments, Z1 is absent and Z2 is a cysteine ​​residue. In some embodiments, at least one of Z1 and Z2 is present. In some embodiments, Z2 is absent and Z1 is a cysteine ​​residue. In some embodiments, Z1 is a cysteine ​​residue and Z2 is a cysteine ​​residue.

[0133] In certain embodiments, the peptide compound has the sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 47) or acetyl-GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 48), which corresponds to SEQ ID NO: 10 and SEQ ID NO: 15, modified by the addition of a cysteine ​​residue at the C-terminus.

[0134] The conjugate compound can include, for example, 1 to 10 or 1 to 5 (e.g., 1, 2, 3, or 4) therapeutic agent (e.g., anti-tumor agent) molecules attached. These therapeutic agent molecules can be the same or different; for example, 2, 3, 4, or more different therapeutic agents can be attached to the peptide compound. The therapeutic agent is attached to the peptide compound via one or more covalent bonds, at least one atom, or at least one linker. In some embodiments, at least two therapeutic agent molecules are attached to A. In some embodiments, at least two molecules are molecules of the same therapeutic agent, e.g., a chemotherapeutic agent.

[0135] Antitumor agents can be any compound capable of inhibiting the growth of and / or killing tumor cells, including, for example, small molecules, peptides, proteins, oligonucleotides (e.g., siRNA, shRNA), radionuclides, antibodies, and drug delivery systems including nanoparticles, liposomes, nanotubes, and graphene particles loaded with therapeutic antitumor agents.

[0136] In one embodiment, the anti-tumor agent is a chemotherapeutic agent. The term "chemotherapeutic agent" refers to an agent that kills tumor cells and / or inhibits their proliferation / growth. Examples of chemotherapeutic agents include alkylating agents (e.g., cyclophosphamide, ifosfamide, mechloretine, chlorambucil, melphalan, dacarbazine, nitrosoureas, temozolomide, carmustine, lomustine, streptozocin, busulfan, procarbazine), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), cytoskeletal disrupting agents (e.g., paclitaxel, docetaxel, abraxane, taxotere, cabazitaxel, etc.), and the like. taxanes), histone deacetylase inhibitors (e.g., vorinostat, romidepsin), topoisomerase I inhibitors (e.g., irinotecan, topotecan), topoisomerase II inhibitors (e.g., etoposide, teniposide, tafluposide), kinase inhibitors (e.g., bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, bizmodegib, dasatinib, nilotinib, osimertinib, crizotinib, dabrafenib, vemurafenib, trametinib, ibrutinib), nucleotide analogs, Precursor analogs (e.g., azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil (5-FU), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, thioguanine), peptide antibiotics (e.g., bleomycin, actinomycin), platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin), retinoids (tretinoin, alitretinoin, bexarotene), vinca alkaloids and their derivatives (e.g., vinblastine, Mitotic inhibitors such as vincristine, vindesine, vinorelbine), toxins such as maytansinoids, auristatins, calicheamicins, amatoxins or amanitin, and natural phytochemicals with antitumor properties, such as curcumin, alkaloids (e.g., chlorogenic acid, theobromine, theophylline), anthocyanins (e.g., cyanidin, malvidin), carotenoids (beta-carotene, lutein, lycopene), coumestane, flavan-3-ols, flavonoids (e.g., epicatechin, hesperidin,Isorhamnetin, kaempferol, myricetin, naringin, nobiletin, proanthocyanidins, quercetin, rutin, tangerine), hydroxycinnamic acids (e.g., chicoric acid, coumarin, ferulic acid, scopoletin), isoflavones (e.g., daidzein, genistein), lignans (e.g., silymarin), monoterpenes (e.g., geraniol, limonene), organosulfides (e.g., allicin, glutathione, indole-3-carbinol, isothiocyanates) , sulforaphane), damanacanthan, digoxin, phytic acid, phenolic acids (e.g., capsaicin, ellagic acid, galactic acid, rosemarinic acid, tannic acid), phytosterols (e.g., beta-sitosterol), saponins, stilbenes (e.g., piterostilbene, resveratrol), triterpenoids (e.g., ursolic acid), xanthophylls (e.g., astaxanthin, beta-cryptoxanthin), monophenols (e.g., hydroxytyrosol),

[0137] In another embodiment, the anti-tumor agent is an antibody or antigen-binding fragment thereof that recognizes an antigen expressed by tumor cells.

[0138] In embodiments, B is attached to A at the free amine of a lysine residue of the peptidic compound, optionally via a linker, or at the N-terminal position of the peptidic compound, optionally via a linker. In embodiments, B is attached to A (optionally via a linker) at a cysteine ​​residue added to the terminus, e.g., the C-terminus, of the peptidic compound.

[0139] In embodiments, B is linked to A via a linker, optionally via a cleavable linker.

[0140] The term "linker" as used herein refers to a chemical structure that connects the peptide compounds disclosed herein to at least one therapeutic agent. The linker can be attached to different functional groups of the peptide compound. For example, the linker can be attached to the primary amine (amine (-NH)) of the peptide compound, which is present at the N-terminus of each polypeptide chain (called the alpha amine) and in the side chain of lysine (Lys, K) residues (called the epsilon amine). For example, the linker can be attached to the peptide compound at the carboxyl (-COOH) group, which is present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). For example, the linker can be attached to the peptide compound at the sulfur hydrogen group (-SH), which is present in the side chain of cysteine ​​(Cys, C). Often, as part of the secondary or tertiary structure of a protein, cysteines are linked between their side chains via disulfide bonds (-SS-). These need to be reduced to sulfur hydrogen groups to make them available for cross-linking by most reactive groups. For example, a linker can be attached to a peptide compound at a carbonyl (-CHO) and a ketone or aldehyde group can be created on the glycoprotein by oxidation of the polysaccharide post-translation modification (glycosylation) with sodium sulfite.

[0141] The table below summarizes the reactivity classes and chemical groups of some major linkers for standard chemical conjugation. [Table 1]

[0142] For example, homobifunctional and heterobifunctional crosslinkers can be used. For example, disuccinimidyl suberate (DSS) is a homobifunctional crosslinker with identical amine-reactive NHS ester groups at both ends of a short spacer arm. For example, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) is a heterobifunctional crosslinker with an amine-reactive sulfo-NHS ester group at one end of a cyclohexane spacer arm and a sulfo-reactive maleimide group at the other end. This allows for a sequential two-step conjugation procedure. Commercially available homobifunctional crosslinkers include BSOCOES (bis(2-[succinimidoxycarbonyloxy]ethyl) sulfone), DPDPB (1,4-di-(3'-[2-pyridylthio]-propionamido)butane), DSS (disuccinimidyl subrate), DST (disuccinimidyl tartrate), SulfoDST (sulfodisuccinimidyl tartrate), DSP (dithiobis(succinimidyl propionate), DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate), EGS (ethylene glycol bis(succinimidyl succinate), and BASED (bis(β-[4-azidosalicylamido]-ethyl) disulfide).

[0143] The peptide compounds can be attached via a variety of linkers, such as, for example, a thiol group, an amino group (amine), or any suitable reactive group. The linker can be a covalent bond. The linker group can include flexible arms, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms.

[0144] Exemplary linkers include, but are not limited to, pyridine disulfide, thiosulfonic acid, vinyl sulfonic acid, isocyanate, imidoester, diazine, hydrazine, thiol, carboxylic acid, multi-peptide linker, and acetylene. Other linkers that can be used include BS 3These include (bis(sulfosuccinimidyl)suberate) (a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS / EDC (N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS / EDC allows for the coupling of primary amine and carboxyl groups), sulfo-EMCS ([N-ε-maleimidocaproic acid]hydrazine (sulfo-EMCS is a heterobifunctional reactive group reactive toward thiols and amino groups), and hydrazine (most proteins contain exposed carbohydrates, and hydrazine is a useful reagent for coupling carboxyl groups to primary amines).

[0145] A wide range of activated carboxyl groups (e.g., esters) can be used as chemically reactive groups to form covalent bonds, and hydroxyl groups can be physiologically tolerated at the required level to modify the peptide compound. Specific reagents include, for example, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), maleimido-benzoyl-succinimide (MBS), gamma-maleimidobutyryloxysuccinimide ester (GMBS), maleimidopropionic acid (MPA), maleimidohexanoic acid (MHA), and maleimidoundecanoic acid (MUA).

[0146] Primary amines are the primary targets of NHS esters, which react with primary amines to form covalent amide bonds. Accessible α-amine groups present at the N-terminus of proteins and the ε-amine of lysine react with NHS esters. Thus, the conjugated compounds disclosed herein include linkers with NHS esters attached to the N-terminal amino acid or ε-amine of lysine of a peptide compound. An amide bond is formed when the NHS ester reacts with a primary amine, releasing an N-hydroxysuxanimide. A suximinyl group containing a reactive group may be more simply referred to as a suximinyl group. In some embodiments, the functional group of the peptide compound is a thiol group, and the chemically reactive group is a maleimide-containing group, such as γ-maleimide-butyramide (GMBA or MPA). Such maleimide-containing groups may be referred to herein as maleido groups.

[0147] Interamine linkers include NHS esters, imidoesters, etc., examples of which are shown below. [Table 2]

[0148] The linker can be an inter-sulfhydryl linker such as maleimide or pyridyldithiol, as shown below. [Table 3]

[0149] The linker can be an amine-sulfhydryl linker, including NHS ester / maleimide compounds. Examples of these compounds are shown below. [Table 4]

[0150] The linker can react with amino groups and non-selective entities. Such linkers include NHS ester / aryl azide and NHS ester / diazirine linkers, examples of which are shown below. [Table 5]

[0151] Exemplary amine-carboxyl linkers include carbodiimide compounds (e.g., DCC (N,N-dicyclohexylcarbodiimide) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide)). Exemplary sulfhydryl-nonselective linkers include pyridyldithiol / allyl azide compounds (e.g., APDP (N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide)). Exemplary sulfhydryl-carbohydrate linkers include maleimide / hydrazide compounds (e.g., BMPH (N-[β-maleimidopropionic acid]hydrazide), EMCH ([N-ε-maleimidocarbohydrate]hydrazide), MPBH4-(4-N-maleimidophenyl)butyric acid hydrazide), and KMUH (N-[κ-maleimidoundecanoic acid]hydrazide)) and pyridyldithiol / hydrazide compounds (e.g., PDPH (3-(2-pyridyldithio)propionylhydrazide)). Exemplary carbohydrate-nonselective linkers include hydrazide / aryl azide compounds (e.g., ABH (p-azidobenzoylhydrazide)). Exemplary hydroxyl-sulfhydryl linkers include isocyanate / maleimide compounds (e.g., (N-[p-maleimidophenyl]isocyanate)). Exemplary amine-DNA linkers include NHS ester / psoralen compounds (eg, SPB (suxinimidyl-[4-(psoralen-8-yloxy)]-butyrate)).

[0152] In the conjugated peptide compound, the linker can connect 3 to 7 entities to generate branching points of different complexity. [Table 6]

[0153] TMEA and TSAT have access to sulfhydryl groups through their respective maleimide groups. The hydroxy and carboxy groups of THPP can react with primary or secondary amines. Other useful linkers follow the formula Y=C=NQAC(O)-Z, where Q is a homoaromatic or heteroaromatic ring system and A is a single bond or an unsubstituted or substituted divalent C 1-30 a bridging group, Y is O or S, Z is Cl, Br, I, N, N-succinimidyloxy, imidazolyl, 1-benzotriazolyloxy, OAr (where Ar is an electron-deficient activated aryl group), or OC(O)R (where R is -AQN=C=Y or C 4-20 tertiary alkyl) (see U.S. Pat. No. 4,680,338).

[0154] Other useful linkers are [ka] and R1 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 6-12 aryl or aralkyl, or divalent organic -O-, -S-, or [ka] and R' is C 1-6 The linking moiety of the alkyl, R2 is H, C 1-12 Alkyl, C 6-12 Aryl, or C 6-12 aralkyl, and R3 is [ka] [ka] [ka] [ka] [ka] [ka] [ka] or another chemical structure capable of delocalizing the lone pair of electrons of the adjacent nitrogen, and R4 is a reactive pendant group capable of attaching R3 to a peptide compound (see, e.g., U.S. Pat. No. 5,306,809).

[0155] The linker can comprise at least one amino acid residue and can be a peptide of at least or about 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, or 50 amino acid residues. When the linker is a single amino acid residue, it can be a naturally occurring or non-naturally occurring amino acid (e.g., Gly or Cys). When the linker is a short peptide, it is preferred to use a glycine-rich peptide (e.g., the sequence [Gly-Gly-Gly-Gly-Ser]) (which tend to be flexible). n where n is an integer between 1 and 6, inclusive (see U.S. Pat. No. 7,271,149) or a serine-rich peptide linker (see U.S. Pat. No. 5,525,491). A serine-rich peptide linker can be a peptide having the formula [XXXX-Gly] y wherein up to two of X are Thr and the remaining X are Ser, and y is an integer greater than 1, for example, 1 to 5 (inclusive), e.g., [Ser-Ser-Ser-Ser-Gly] y (SEQ ID NO: 19), where y is an integer equal to or greater than 1, e.g., 1 to 5. Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 20) and (PT) n P, where n is 2, 3, 4, 5, 6, or 7) and an α-helical linker (e.g., A(EAAAK) n A (SEQ ID NO: 21), where n is 1, 2, 3, 4, or 5).

[0156] The linker can be an alkyl linker (e.g., amide-linked to the polypeptide and ester-linked to the therapeutic agent). If an aliphatic linker is used, it can be of any length (e.g., C1-C 20 , C1-C 12 , C1-C6) and the chemical functional groups they contain (e.g., amino groups or carbamates).

[0157] Examples of suitable amino acid linkers include succinic acid, lysine, glutamic acid, aspartic acid, or the dipeptide glycine-lysine. When the linker is succinic acid, one of its carboxyl groups can form an amide bond with the amino group of the amino acid residue, and the other carboxyl group can form an amide bond with, for example, the amino group of a peptide or a substituent. When the linker is lysine, glutamic acid, or aspartic acid, its carboxyl group can form an amide bond with the amino group of the amino acid residue, and the amino group can form an amide bond with, for example, the carboxyl group of a substituent. When lysine is used as a linker, an additional linker can be inserted between the ε-amino group of lysine and the substituent. The additional linker can be succinic acid, which can form an amide bond between the ε-amino group of lysine and the amino group present in the substituent. In one embodiment, the additional linker is glutamic acid or aspartic acid (e.g., forms an amide bond with the ε-amino group of lysine and another amide bond with the carboxyl group present in the substituent), i.e., the substituent is N-amino. ε -acylated lysine residues.

[0158] The linker can also be a branched polypeptide. Exemplary branched peptide linkers are described in U.S. Patent No. 6,759,509.

[0159] The linker can provide a cleavable bond (e.g., a thioester bond) or a non-cleavable bond (e.g., a maleimide bond). For example, a cytotoxic protein can be attached to a linker that reacts with a lysine residue in a polypeptide or a modified free amine present at the amino terminus of a polypeptide. Thus, linkers useful in the present conjugates can contain a group reactive with a primary amine of the polypeptide or modified polypeptide to which the therapeutic moiety is attached. More specifically, the linker can be selected from monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-S-acetylthiopropionic acid (SATP), maleimide, and dibenzocyclooctyne esters (DBCO esters). Useful cyclooctynes ​​within specific linkers include OCT, ALO, MOFO, DIFO, DIBO, BARAC, DIBAC, and DIMAC.

[0160] The linker can include a flexible arm, such as a short arm (<2 carbon chain), a medium sized arm (2-5 carbon chain), or a long arm (3-6 carbon chain).

[0161] Click chemistry can also be used for conjugation on peptides (DBCO, TCO, tetrazine, azide, and alkyne linkers). This family of linkers can be reactive to amine, carboxyl, and sulfhydryl groups. Furthermore, these linkers can be biotinylated, PEGylated, modified with fluorescent imaging dyes, or phosphoramidated for incorporation into oligonucleotide sequences.

[0162] In embodiments, the anti-tumor agent-peptide compound conjugate is represented by formula (LIII) or (LIV): GVRAK(J 1 )AGVRN(Nle)FK(J 2 ) SESY(LIII) (SEQ ID NO: 22), Acetyl-GVRAK(J 1 )AGVRN(Nle)FK(J2 ) SESY(LIV) (SEQ ID NO: 23), J 1 and J 2 are each independently an anti-tumor (e.g., chemotherapeutic) agent attached to a lysine (K) residue.

[0163] In one embodiment, the conjugate compound is GVRAK(curcumin)AGVRN(Nle)FK(curcumin)SESY-Formula (XIV) (SEQ ID NO: 24), which comprises a peptide compound having SEQ ID NO: 10, wherein each lysine residue has a curcumin molecule bound thereto, or YK(curcumin)SLRRK(curcumin)APRWDAPLRDPALRQLL-Formula (XV) (SEQ ID NO: 25), which comprises a peptide compound having SEQ ID NO: 11, wherein each lysine residue has a curcumin molecule bound thereto.

[0164] In one embodiment, the conjugate compound is acetyl-GVRAK(curcumin)AGVRN(Nle)FK(curcumin)SESY-Formula (XVI) (SEQ ID NO: 26), which comprises a peptide compound having SEQ ID NO: 15, wherein each lysine residue has a curcumin molecule bound thereto, or acetyl-YK(curcumin)SLRRK(curcumin)APRWDAPLRDPALRQLL-Formula (XVII) (SEQ ID NO: 27), which comprises a peptide compound having SEQ ID NO: 16, wherein each lysine residue has a curcumin molecule bound thereto.

[0165] In one embodiment, the conjugate compound is GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY-Formula (XIX) (SEQ ID NO: 28), including a peptide compound having SEQ ID NO: 10, wherein each lysine residue has a docetaxel molecule bound to it.

[0166] In another embodiment, the conjugate compound is Acetyl-GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY-Formula (XXIII) (SEQ ID NO: 29), which comprises a peptide compound having SEQ ID NO: 15, wherein each lysine residue has a docetaxel molecule bound to it.

[0167] In certain embodiments, the conjugate compound is GVRAK(doxorubicin)AGVRN(Nle)FK(doxorubicin)SESY-Formula (XXVI) (SEQ ID NO: 30), which comprises a peptide compound having SEQ ID NO: 10, wherein each lysine residue has a doxorubicin molecule attached thereto.

[0168] In another embodiment, the conjugate compound is acetyl-GVRAK(doxorubicin)AGVRN(neridine)FK(doxorubicin)SESY-formula (XXVIII) (SEQ ID NO: 31), comprising a peptide compound having SEQ ID NO: 15, wherein each lysine residue has a doxorubicin molecule attached thereto.

[0169] In some embodiments, the conjugate compound comprises a peptide compound having SEQ ID NO: 47 (wherein a cysteine ​​residue has an aldoxorubicin molecule attached thereto), or a peptide compound having SEQ ID NO: 10 (wherein a cysteine ​​residue is added to the C-terminus of the peptide compound and the cysteine ​​residue has an aldoxorubicin molecule attached thereto), GVRAKAGVRN(Nle)FKSESYC(aldoxorubicin)-Formula (LI) (SEQ ID NO: 32).

[0170] In some embodiments, the conjugate compound comprises a peptide compound having SEQ ID NO: 48 (wherein a cysteine ​​residue has an aldoxorubicin molecule attached thereto), or a peptide compound having SEQ ID NO: 15 (wherein a cysteine ​​residue is added to the C-terminus of the peptide compound and the cysteine ​​residue has an aldoxorubicin molecule attached thereto), acetyl-GVRAKAGVRN(Nle)FKSESYC(aldoxorubicin)-Formula (LII) (SEQ ID NO: 33).

[0171] In some embodiments, the conjugates are administered in the form of a prodrug. As used herein, the term "prodrug" refers to a derivative of an active form of a known compound or composition that, when administered to a subject, is gradually converted to an active form, resulting in a better therapeutic response and / or reduced toxicity levels. Generally, prodrugs are functional derivatives of the compounds disclosed herein and are readily convertible in vivo to the conceptually derived compound. Prodrugs include, but are not limited to, acyl esters, carbonates, phosphates, and ureas. These groups are exemplary and not exhaustive, and one of skill in the art can prepare other known prodrug variants. Prodrugs can be formed, for example, using available hydroxy, thiol, amino, or carboxy groups. For example, available OH and / or NH groups of the disclosed conjugates can be acylated with an activated acid in the presence of a base, optionally also in an inert solvent (e.g., an acid chloride in pyridine). Common esters utilized as prodrugs include phenyl esters, aliphatic (C1-C2) esters, and esters of phenyl esters. 24 ) esters, acyloxymethyl esters, carbamates, and amino acid esters. Under certain circumstances, prodrugs of the compounds of the present disclosure are those in which hydroxy and / or amino groups in the compounds are masked as groups that can be converted to hydroxy and / or amino groups in the body. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0172] Covalent modifications of conjugates are included within the scope of this disclosure. Covalent modifications include reacting the targeted amino acid residue of the conjugate with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues of the conjugate. Other modifications include deamidation of glutamic acid and aspartic acid residues to the corresponding glutamic acid and aspartic acid residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, and methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)). Other types of covalent modifications of conjugates within the scope of this disclosure include coupling the conjugate to proteins (e.g., albumin) or nonproteinaceous polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes), which may increase the in vivo half-life of the conjugate.

[0173] In some embodiments, the conjugate compounds disclosed herein, or pharmaceutically acceptable salts thereof, are prepared into pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Such compositions can be prepared by methods well known in the pharmaceutical arts by mixing a conjugate compound of appropriate purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr., 2012, 22). nd edition,Pharmaceutical Press;Handbook of Pharmaceutical Excipients,by Rowe et al.,2012,7 th(See, e.g., 1999 edition, Pharmaceutical Press). The carrier / excipient is suitable for administration of the conjugate compound by any conventional route of administration, such as oral, intravenous, parental, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intracerebroventricular, intracapsular, intraspinal, intramedullary, epidural, intraventricular, intraperitoneal, intranasal, or pulmonary (e.g., aerosol) administration. In certain embodiments, the carrier / excipient is adapted for administration of the conjugate compound or a salt thereof by an intravenous or subcutaneous route. In certain embodiments, the carrier / excipient is adapted for administration of the conjugate compound by an intravenous route. In other embodiments, the carrier / excipient is adapted for administration of the conjugate compound or a salt thereof by a subcutaneous route. In other embodiments, the carrier / excipient is adapted for administration of the conjugate compound or a salt thereof by an oral route.

[0174] As used herein, the term "excipient" has its usual meaning in the art and refers to any ingredient that is not the active ingredient (drug) itself. Excipients include, for example, binders, lubricants, diluents, fillers, thickeners, disintegrants, plasticizers, coating agents, barrier layer formulations, lubricants, stabilizers, release retardants, and other ingredients. As used herein, a "pharmaceutically acceptable excipient" refers to an excipient that does not interfere with the effectiveness of the biological activity of the active ingredient and is not toxic to the subject, i.e., a type of excipient, and / or any excipient for use in an amount that is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, the composition may contain excipients, including one or more binders, thickeners, surfactants, diluents, release retardants, colorants, flavoring agents, fillers, disintegrants / solubility enhancers, lubricants, plasticizers, silica flow modifiers, glidants, anti-caking agents, anti-adherents, stabilizers, antistatic agents, swelling agents, and any combination thereof. As those skilled in the art will recognize, a single excipient can perform more than one function at a time, for example, acting as both a binder and a thickener. Furthermore, as those skilled in the art will recognize, these terms are not necessarily mutually exclusive. Examples of excipients commonly used for injectable solutions include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. It is often preferable to include an isotonicity agent, e.g., a sugar, a polyhydric alcohol such as mannitol or sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances include auxiliary substances such as wetting agents, emulsifying agents, preservatives, and buffers that increase shelf life or effectiveness.

[0175] The precise amount / dose of the conjugate administered will vary depending on the specific cancer cells and the specific cancer disease involved, the extent, involvement, or severity of the cancer disease, the size, age, and general health of the cancer patient, the individual patient's response, the specific compound administered, the bioavailability characteristics of the administered formulation, the selected administration schedule, whether the conjugate is administered alone or in combination with other drugs, the pharmacodynamic properties of the conjugate and its method and route of administration, and other relevant characteristics that a physician or skilled artisan can readily determine using known techniques and by observing results obtained under similar circumstances. The conjugate / composition is suitably administered to the patient at one time or over a series of treatments. Preferably, a dose-response curve is determined in vivo, followed by useful animal models prior to human testing. The present disclosure provides dosages for these conjugates and compositions containing them. For example, doses ranging from about 1 μg per kilogram of body weight (μg / kg) to 1000 mg per kg of body weight (mg / kg) are administered daily, depending on the type and severity of the disease. Furthermore, effective dosages range from 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, in 25 mg / kg increments up to 1000 mg / kg, and may vary between any two of the above values. Typical daily dosages range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, treatment is continued until a desired suppression of disease symptoms occurs, depending on the condition. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0176] The conjugate compounds described herein or salts thereof, or compositions comprising same, can be used in combination with one or more additional agents or therapies (e.g., radiation therapy, surgery, vaccines, etc.) for the treatment of a targeted disease / condition or for the management of one or more symptoms of a targeted disease / condition (e.g., pain relievers, anti-nausea agents, etc.). In certain embodiments, the conjugate compounds described herein are used in combination with one or more chemotherapeutic agents, immunotherapies, checkpoint inhibitors, cell-based therapies, etc. Examples of chemotherapeutic agents suitable for use in combination with the conjugates described herein include, but are not limited to, vinca alkaloids, agents that disrupt microtubule formation (such as colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR-targeted agents, tyrosine kinase-targeted agents (such as tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, metabolic inhibitors (such as nucleoside analogs), alkylating agents, platinum compounds, anthocycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acid or its derivatives), geldanamycin or its derivatives (such as 17-AAG), and other art-recognized cancer therapeutics.In some embodiments, chemotherapeutic agents for use in combination with the conjugates described herein include one or more of adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmostin (BCNU), methyl-CCNU, cisplatin, etoposide, interferon, camptothecin and its derivatives, phenesterine, taxanes and its derivatives (e.g., taxol, paclitaxel and its derivatives), and the like. and derivatives thereof, docetaxel and its derivatives), topotecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ortataxel, gemcitabine, oxaliplatin, Herceptin®, vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®, Tarceva®, Neuresta®, lapatinib, sorafenib, erlotinib, Erbitux and its derivatives, etc. In some embodiments, the conjugate compounds described herein or compositions comprising same are used in combination with an EGFR or tyrosine kinase targeted therapy, for example, an EGFR inhibitor (RTK inhibitor). The conjugate compounds described herein or salts thereof, or compositions comprising same, may be used in combination with one or more therapeutic antibodies or antibody fragments, such as, for example, therapeutic antibodies or antibody fragments used in the treatment of tumors.Examples of antibodies used in cancer therapy include antibodies targeting CD52 (e.g., alemtuzumab), VEGF / VEGFR (e.g., bevacizumab, lamisilumab), EGFR (e.g., cetuximab, necituzumab, panitumumab), CD38 (e.g., daratumumab, isatuximab), RANKL (e.g., denosumab), GD2 (e.g., dinutuximab, naxitamab-gqgk), SLAMF7 (e.g., elotuzumab), HER2 (e.g., margetuximab-cmkb, pertuzumab), CCR4 (e.g., mogamulizumab), CD20 (obinutuzumab), and the like. , ofatumumab, rituximab), BCMA (e.g., teclistamab), CD19 (e.g., tafasitamab), CTLA-4 (e.g., tremelimumab), LAG-3 (e.g., leratilimumab), PD-1 (e.g., tislelizumab, pemprylizumab, sintilimab, toripalimumab, retifanlimab, dostarimab), PD-L1 (e.g., darvalumab, avelumab, atezolizumab), EpCAM (e.g., oporutumab, edrecolomab), nectin-4 (e.g., enfortumab), CD79b (e.g., polatuzumab).

[0177] In one embodiment, the conjugate compound defined herein or a salt thereof is used in combination with immunotherapy (eg, an immunotherapeutic agent).

[0178] Thus, in another aspect, the present disclosure provides a method of treating cancer comprising administering to a subject a therapeutically effective amount of a peptide compound or conjugate as defined herein in combination with immunotherapy (e.g., immune checkpoint inhibitor therapy). The present disclosure also provides the use of a peptide compound or conjugate as defined herein in combination with immunotherapy (e.g., immune checkpoint inhibitor therapy) for the treatment of cancer or for the manufacture of a medicament for the treatment of cancer. The present disclosure also provides combination therapies comprising a peptide compound or conjugate as defined herein and immunotherapy (e.g., immune checkpoint inhibitor therapy) for use in the treatment of cancer.

[0179] In certain embodiments, the peptide compound or conjugate and the immunotherapy exhibit a synergistic effect (eg, tumor growth inhibition, survival rate, etc.) in a subject.

[0180] As used herein, the term "immunotherapy" refers to antitumor treatments that strengthen or improve the immune response against tumor cells. Immunotherapy includes cell-based immunotherapy, which involves the administration of immune cells that can recognize tumor cells, such as chimeric antigen receptor (CAR) T cells or NK cells, T cells with tumor antigen-specific TCRs, or antigen-presenting cells (APCs, such as dendritic cells) that can express tumor antigens on their surface. Immunotherapy also includes the administration of specific antibodies that recognize antigens expressed by tumor cells and target them for destruction by the immune system, or the administration of cytokines (interferons, interleukins) that stimulate the immune response. Another type of immunotherapy involves the administration of immune checkpoint inhibitors (ICIs). Combinations of different types of immunotherapy may also be used, such as the administration of immune cells (CAR T or NK cells) in combination with immune checkpoint inhibitors.

[0181] As used herein, the term "immune checkpoint inhibitor" (ICI) or "immune checkpoint blocker" (ICB) refers to an agent that blocks or suppresses the activity of negative regulators of the immune response. In one embodiment, the ICI blocks or suppresses the activity of T cells (e.g., CTL and / or CD4 helper T cells) and / or NK cells. Examples of negative regulators of the immune response (i.e., immune checkpoints) include adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), inhibitory factor B and T lymphocytes (BTLA or CD272), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, CD152), CD47 / SIRPα, indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), poliovirus receptor-related immunoglobulin (PVRIG), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), programmed death 1 (PD-1) receptor, PD-L1, PD-L2, T-cell immunoglobulin domain and mucin-like domain 3 (TIM-3), V-domain Ig inhibitor of T-cell activation (VISTA), and sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7 or CD328) and SIGLEC9 (CD329). In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, or PD-L1. Examples of immune checkpoint inhibitors include anti-PD-1 antibodies / blockers (e.g., tislelizumab, penprimab, pidilizumab, sintilimab, toripalimab, retifanlimab, dostallimab, nivolumab, cemiplimab, pembrolizumab, spartalizumab, camrelizumab, JTX-4014, INCMGA00012 (MGA012), AMP-224, A MP-514), anti-PD-L1 antibodies / blockers (e.g., darvalumab, avelumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189), anti-CTLA-4 antibodies (e.g., tremelimumab, ipilimumab), anti-LAG-3 antibodies (e.g., leratilimab, LAG525 (IMP701), REGN3767 (R3767), BI 754,091, tebotelimab (MGD013), eftiradimod alfa (IMP321, FS118), anti-TIM-3 antibodies (MBG453, Sym023, TSR-022), anti-B7-H3 / H4 antibodies (e.g., MGC018, FPA150), adenosine signaling pathway inhibitors including A2AR inhibitors (e.g., inupadenant (EOS100850), etormadenant (AB928), imaradenant (AZD4635), ciforadenant, NIR178, CS3005, PBF-999, INCB106385, CPI-444), CD73 antagonists / anti-CD73 antibodies (e.g., mupadrimab (CPI-006 ), oleculab (MEDI9447), uriledolimab, AB680, BMS-986179, NZV930, AK119, SYM024, INCA00186, ORIC-533, IPH5301, PSB-1248937), and CD39 antagonists (TTX-030, IPH5201, SRF617), anti-NKG2A antibodies (monalizumab), anti-PVRIG (e.g., CyOM701), anti-CEACAM1 antibodies (e.g., CM24), and CD47 blockers / inhibitors (evoluptuous cycle inhibitors (ALX148), Hu5F9-G4 (5F9), TTI-662, RRx-001) (e.g., Marin-Acevedo See, e.g., Xia et al., "Next generation of immune checkpoint inhibitors and beyond," Journal of Hematology & Oncology, volume 14, Article number: 45 (2021); Xia et al., "CD39 / CD73 / A2AR pathway and cancer immunotherapy," Molecular Cancer, volume 22, Article number: 44 (2023)." The chemical structures and sequences of the above immune checkpoint inhibitors are incorporated herein by reference.

[0182] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1, such as an anti-PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, a combination of ICIs is used, such as a combination of a CTLA-4 inhibitor and a PD-1 inhibitor (e.g., ipilimumab and nivolumab) or a combination of a CTLA-4 inhibitor and a PD-L1 inhibitor (e.g., darvalumab and tremelimumab).

[0183] The combination of active agents (e.g., conjugate compound + immunotherapeutic agent) and / or compositions comprising same can be administered in any conventional dosage form or co-administered (e.g., sequentially, simultaneously, at different times). Co-administration in the context of this invention refers to the administration of two or more therapies in a course of treatment coordinated to achieve an improved clinical outcome. Such co-administration can also occur during overlapping time periods, i.e., coexisting. For example, a first agent (e.g., a conjugate compound described herein) can be administered to a patient before, simultaneously with, before, after, or after the administration of a second active agent (e.g., a chemotherapeutic agent or immunotherapy). The agents, in some embodiments, can be combined and prepared in a single composition and thus administered simultaneously.

[0184] In one embodiment, enhancing the anti-tumor immune response comprises (a) increasing the number of tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and / or natural killer (NK) cells within the tumor, and / or (b) decreasing the level of immunoregulatory cells within the tumor. In one embodiment, TILs are activated and / or memory CD4 + and / or CD8 + T cells, e.g. cytotoxic CD8 +In some embodiments, the TAMs comprise type 1 macrophages (M1). In some embodiments, the NK cells comprise cytotoxic NK cells. In some embodiments, the immunoregulatory cells are CD4 + These include regulatory T cells (Tregs), type 2 macrophages (M2), and / or NK regulatory cells (NKregs).

[0185] The cancer can be any type of cancer, including primary (or original), recurrent, or metastatic cancer. Examples of cancer include cardiac sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated giant cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma (e.g., Ewing's sarcoma, Kaposi's sarcoma), lymphoma, chondroitinous hamartoma, mesothelioma; cancers of the gastrointestinal system, such as esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreatic (tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), cancer of the genitourinary tract, e.g., kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma) , choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma; liver cancer, e.g., hepatoma (hepatocellular carcinoma, HCC), intrahepatic bile duct carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (e.g., pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor, and glucagonoma); bone cancer, e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma chronfroma) (osteochondral exostoses), benign chordoma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor; cancers of the nervous system, e.g., neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, embryonal tumor [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibroma, meningioma, glioma, sarcoma);Cancers of the reproductive system, such as gynecological cancers, uterine cancer (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovarian cancer (ovarian carcinoma [serous pancreatic cystadenoma, mucinous cystadenocarcinoma, undifferentiated carcinoma], granulosa theca cell tumor, Sertoli-Leydegg cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube cancer (carcinoma); placental cancer, penile cancer, prostate cancer , testicular cancer; cancers of the blood system, e.g., blood cancers (acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; cancers of the oral cavity, e.g., lip cancer, tongue cancer, gum cancer, palate cancer, oropharynx cancer, nasopharyngeal cancer, maxillary sinus cancer; skin cancer, e.g., malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentiginous dysplastic nevus (moles dysplastic neoplasms, lipomas, hemangiomas, dermatofibromas, and keloids; adrenal cancer; neuroblastoma; and cancers of other tissues, including connective and soft tissues, retroperitoneal cavity, and peritoneum, eye cancer, ocular melanoma, and adnexal cancer, breast cancer (e.g., ductal carcinoma), head and / or neck cancer (head and neck squamous cell carcinoma), anal cancer, thyroid cancer, parathyroid cancer; secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites;

[0186] In some embodiments, the cancer is an immunologically cold (or ignorant) cancer. The term "immunologically cold cancer" or "cold cancer" refers to a cancer that does not elicit an anti-tumor immune response in patients and / or does not respond to cancer immunotherapy, such as ICI therapy (see Bonaventura et al., "Cold Tumors: A Therapeutic Challenge for Immunotherapy," Frontiers in Immunology, vol. 10, pp. 168 (2019)). Immunologically cold cancers are characterized by the absence of infiltration of anti-tumor immune cells, such as TILs, TAMs, and / or NK cells, and / or the presence of high levels of immune regulatory cells (e.g., Tregs) within the tumor. Immunologically cold tumors can be subdivided into so-called immune wilderness tumors, in which immune effector cells, such as T cells, are absent from or around the tumor, and immune-excluded tumors, in which immune effector cells, such as T cells, accumulate near the tumor but do not effectively infiltrate it. In some embodiments, immunologically cold cancers are immune wilderness tumors. In another embodiment, the immunologically cold cancer is an immunoexcluded cancer.

[0187] Some breast, ovarian, prostate, pancreatic, and glioma cancers are considered immunologically cold cancers. Several cancer subtypes are also immunologically cold, including lung cancer subtypes such as non-small cell lung cancer (NSCLC) (Cascone et al., Tumor Immunology and Immunotherapy of Non-Small-Cell Lung Cancer, Cold Spring Harb Perspect Med. 2022 May 27;12(5):a037895), renal cancers such as pigment-oriented renal cell carcinoma (nccRCC) (Zarrabi et al., Immune Checkpoint Inhibition in Advanced Non-Clear Cell Renal Cell Carcinoma: Leveraging Success from Clear Cell Histology into New Opportunities, Cancers vol. 13,15 3652. 21 Jul. 2021), and renal cell carcinoma with a high tumor mutation burden (Yakirevich et al., Tumor mutational burden and immune signatures interplay in renal cell carcinoma. Ann Transl Med. 2020;8(6):269), colorectal cancer (CRC) including consensus molecular subtype (CMS) 2 and CMS 3 CRC (Roelands et al., Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications, Int J Mol Sci. 2017;18(10):2229), head and neck squamous cell carcinoma (Ribbat-Idel et al., Immunologic “Cold” Squamous Cell Carcinomas of the Head and Neck Are Associated With an Unfavorable Prognosis, Frontiers in medicine, 8,622330), and esophageal cancer (Puhr et al., Immunotherapy for Esophageal Cancers: What Is Practice Changing in 2021?, Cancers vol. 13, 18 4632), and liver cancers such as stage II hepatocellular carcinoma (Nguyen et al., Nature Communications volume 13, Article number: 1441 (2022)). Homozygous deletion of 9p21.3 is one of the most frequent genomic defects in approximately 13% of all cancers, including melanoma (SKCM), bladder cancer (BLCA), pancreatic cancer (pancreatic adenocarcinoma), gastric cancer (gastric adenocarcinoma), lung adenocarcinoma (LUAD), and squamous cell carcinoma (LUSC), and has been shown to be associated with an immunologically cold phenotype.

[0188] In some embodiments, the methods and uses described herein further comprise determining whether a patient is susceptible to (or identifying a patient susceptible to) an immunologically cold cancer. Determining whether a patient is susceptible to an immunologically cold cancer can be done by various methods known in the art. For example, this determination can be made by determining whether a patient is susceptible to an immunologically cold cancer by measuring the number of TILs (cytotoxic CD8 T cells) in the tumor microenvironment (TME). + This may be done by assessing the presence of immune cells such as T cells, TAM (M1), and / or NK cells (cytotoxic NK cells), where an absence or low number of immune cells in the TME indicates that the patient has an immunologically cold cancer, and / or by assessing the presence of immunoregulatory cells (e.g., Tregs) in the TME, where the presence of immunoregulatory cells (e.g., a high number of immunoregulatory cells) (e.g., Tregs) in the TME indicates that the patient has an immunologically cold cancer. A "low number of immune cells" means a number of immune cells that is significantly lower than the average or normal number of immune cells found in a corresponding tumor of the same type, and a "high number of immunoregulatory cells" means a number of immunoregulatory cells that is significantly higher than the average or normal number of immunoregulatory cells (e.g., Tregs) found in a corresponding tumor of the same type.

[0189] In one embodiment, the cancer is resistant to immunotherapy, i.e., refers to a cancer in which immunotherapy does not lead to tumor growth inhibition in the patient. A cancer that is resistant to immunotherapy is either a cancer that has never responded to immunotherapy (primary resistance) or a cancer that has developed resistance to immunotherapy treatment after a period of (responsive) treatment (acquired resistance).

[0190] In a further embodiment, the cancer is resistant to PD-1 or PD-L1 inhibitor-based therapy (anti-PD-1 / PD-L1 therapy). In a further embodiment, the cancer resistant to PD-1 or PD-L1 inhibitor-based therapy is melanoma, lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, colorectal cancer, liver cancer, gastric cancer, squamous cell skin cancer, or multiple myeloma.

[0191] Immune checkpoint inhibitors have been approved for or are currently being tested in phase III and IV clinical trials for several cancers, including lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer, squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal tumors, urothelial carcinoma, colorectal cancer), mesothelioma (e.g., pleural mesothelioma), breast cancer (e.g., triple-negative breast cancer, TNBC), esophageal tumors, multiple myeloma, gastric and esophageal junction cancer, gastric adenocarcinoma, melanoma, Markel cell carcinoma (MCC), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma), liver cancer (e.g., hepatocellular carcinoma), melanoma, ovarian cancer, fallopian tube cancer, peritoneal tumors, bladder cancer, transitional cell carcinoma, prostate tumors, and biliary tract tumors (e.g., Darvin et al., Experimental & Molecular Medicine volume 1, 2014). 50, Article number: 165 (2018). Thus, in one embodiment, the cancer is one of the above cancers for which immune checkpoint inhibitors are approved or currently being tested in phase III and IV clinical trials.

[0192] Currently approved immune checkpoint inhibitors include the anti-CTLA-4 ipilimumab (melanoma and lung cancer), the anti-PD-1 nivolumab (melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colorectal cancer, liver cancer), pembrolizumab (melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, renal cell carcinoma, and gastric cancer), cemiplimab (squamous cell skin cancer, meningioma, lung cancer), and the anti-PD-L1 atezolizumab (non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer), avelumab (non-small cell lung cancer, Merkel cell carcinoma), and darvolimab (urothelial carcinoma, lung cancer). Thus, in certain embodiments, the cancer is one of the above cancers for which immune checkpoint inhibitors are approved.

[0193] In some embodiments, the dosage of a conjugate compound described herein and / or the dosage of an immunotherapy used / administered in a disclosed method, use, composition, or combination therapy is a suboptimal dosage. As used herein, a "suboptimal dosage" refers to a dose of any compound (peptide compound and / or immunotherapy described herein) in a combination described herein that, when used in the absence of the other compounds in the combination, results in a biological effect of 50% or less, in some embodiments 40% or less, in further embodiments 30% or less, in further embodiments 20% or less, and in further embodiments 10% or less. Thus, when a combination of compounds described herein is used in which one or more compounds in the combination are used at a suboptimal dose, a greater efficacy / biological effect may be achieved at the relatively suboptimal dose compared to when the compound is used in the absence of the other compounds.

[0194] As used herein, synergy is achieved when the effect of the combined compounds exceeds the theoretical sum of the effects of each agent in the absence of the other compound. One potential advantage of synergistic combination therapy is the ability to achieve enhanced therapeutic efficacy with reduced toxicity using lower (e.g., suboptimal) doses of one or both agents or therapies. In certain examples, the combination therapy (peptide compounds and / or immunotherapies described herein) provides at least a 5% increase in efficacy over the predicted theoretical additive effect of the agents. In yet another embodiment, the combination therapy provides at least a 10% increase in efficacy over the predicted theoretical additive effect of the agents. In yet another embodiment, the combination therapy provides at least a 20% increase in efficacy over the predicted theoretical additive effect of the agents. In yet another embodiment, the combination therapy provides at least a 30% increase in efficacy over the predicted theoretical additive effect of the agents. In yet another embodiment, the combination therapy provides at least a 50% increase in efficacy over the predicted theoretical additive effect of the agents. An additional benefit of using drug combinations is the potential for efficacy in situations where either drug alone is ineffective, e.g., against cancers or tumors that are resistant to ICIs. Resistance means that administration of an ICI alone does not result in a significant therapeutic benefit, e.g., a significant reduction in tumor volume or tumor cell count, or an increase in survival time. Examples of cancers in which resistance to ICIs has been reported in patients and / or animal models include lung cancer (e.g., NSCLC), pancreatic cancer, prostate cancer, melanoma, ovarian cancer, urothelial carcinoma, and renal cell carcinoma (see, e.g., Fares et al., American Society of Clinical Oncology Educational Book 39, 147-164, 2019; Pandey et al., Cancer Drug Resist 2019;2:178-188). In some embodiments, the synergistic effect is associated with an inhibition or reduction in tumor growth. In some embodiments, the synergistic effect is associated with an increase in survival time.

[0195] Accordingly, in another aspect, the present disclosure provides combination therapies comprising a peptide compound described herein and an immunotherapy, such as immune checkpoint inhibitor (ICI) therapy. The present disclosure also provides the use of combination therapies comprising a peptide compound described herein and an immunotherapy (i.e., an immunotherapeutic agent), such as an ICI, to treat a patient suffering from cancer (e.g., a cancer resistant to immunotherapy, such as ICI monotherapy). The present disclosure also provides the use of combination therapies comprising a peptide compound described herein and an immunotherapy, such as an immune checkpoint inhibitor, for the manufacture of a medicament for treating a subject suffering from cancer resistant to immunotherapy, such as ICI monotherapy. The present disclosure also provides a method of treating a subject suffering from cancer (e.g., a cancer resistant to immunotherapy, such as ICI monotherapy), comprising administering to the subject an effective amount of combination therapy comprising a peptide compound described herein and an immunotherapy, such as an ICI.

[0196] As used herein, the term "subject" or "patient" refers to a mammal, such as a rodent, cat, dog, primate, etc. A subject or patient according to the present disclosure is preferably a human.

[0197] Example The present invention is illustrated in further detail by the following non-limiting examples.

[0198] Example 1: Materials and Methods Cells and reagents. Human SK-MEL-28 melanoma cells containing stage I and II melanosomes were obtained from the American Type Culture Collection (ATCC, Manassas, VA; #HTB-72) [17,18] and cultured in Eagle's Minimum Essential Medium (EMEM; Wisent, #217-010-XK) containing 1 mM Na-pyruvate and 10% fetal bovine serum (FBS; Hyclone, #SH30396.03) at 37°C in a humidified atmosphere (5% CO2). Human A375 melanoma cells were also obtained from ATCC (#CRL-1619) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; #319-005-CL) containing 10% FBS. Human TNBC-derived MDA-MB-231 / Luc cells were obtained from Cell Biolabs, Inc. (San Diego, CA; #AKR-231) and cultured in DMEM containing 10% FBS. Mouse B16-F10 melanoma cells (ATCC; #CRL-6475), a stage III / IV melanoma model [19,20], were cultured in DMEM containing 10% FBS at 37°C in a humidified atmosphere (5% CO2) and used for in vivo syngeneic studies. Cell counting and cell viability were assessed using a BioRad TC20 automated cell counter. A polyclonal anti-SORT1 antibody directed against the 800th amino acid of the C-terminus of the intracellular domain of SORT1 was obtained from Abcam (Cambridge, MA; ab16640). APC anti-mouse CD274 (B7-H1, PD-L1) antibody (#124312) and its APC rat immunoglobulin G (IgG) 2b, kappa isotype control antibody (#400612) were obtained from BioLegend (San Diego, CA). APC major histocompatibility complex class I (MHC-I; H-2Db) monoclonal antibody (mAb; 28-14-8; #17-5999-82) and its APC mouse IgG2a kappa isotype control (eBM2a) (#17-4724-81) were obtained from Thermo Fisher Scientific (Agawam, MA). All other reagents were from Sigma-Aldrich (Oakville, ON).

[0199] Tissue microarray probing and analysis. SORT1 expression was assessed using high-density tissue microarrays (TMA) of human melanoma and healthy tissues at the Institute of Immunology and Cancer Research (IRIC; Montreal, QC). The TMA provided subtype information, allowing for the evaluation of SORT1 expression in different grades of melanoma samples. Immunostaining was performed on 4 μm sections of formalin-fixed, paraffin-embedded material. Briefly, SORT1 antigen retrieval was performed by heat-induced epitope retrieval using ER1 solution (Leica) at 100°C for 30 minutes. Mouse anti-SORT1 (clone F11) primary antibody was incubated at 20 μg / mL for 30 minutes at room temperature. The target antigen was detected using Bond™ polymer purified detection (Leica Biosystems, Buffalo Grove, IL) using diaminobenzidine chromogen for visualization, according to the manufacturer's instructions. Sections were then counterstained with Leica's proprietary hematoxylin and mounted for analysis. Analysis of the images obtained from immunohistochemistry was performed by an expert pathologist. SORT1 labeling was scored using the IHS method on a scale of 0 to 3 as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The raw data were converted to an IHS score by multiplying the quantity and staining intensity scores. Thus, the IHS score ranged from 0 to 12.

[0200] Western blotting Cells were homogenized in 1% sodium dodecyl sulfate (SDS) lysis buffer supplemented with a complete protease inhibitor cocktail from Calbiochem (San Diego, CA). Cells were incubated at room temperature (RT) for 30 min, vortexed every 5 min, sonicated, and then centrifuged at 15,000 g for 10 min at room temperature. Equal amounts of protein (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% nonfat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing 0.1% Tween™-20 (TBST) for 1 h at room temperature. The membranes were washed with TBST and incubated overnight with primary antibodies against SORT1 (1 / 1,000 dilution) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #MAb6C5, 1 / 40,000 dilution) diluted in TBST containing 3% bovine serum albumin (BSA) and 0.05% NaN3. The following antibodies were used: STING (#50494, 1 / 1,000 dilution), p21 (#ab109199, 1 / 1,000 dilution), TANK-binding kinase 1 (TBK1, #3504, 1 / 1,000 dilution), phosphorylated TBK1 (#5483, 1 / 1,000 dilution), p65 (#8242, 1 / 1,000 dilution), phosphorylated p65 (#3033, 1 / 1,000 dilution), and p53 (#ab131442, 1 / 5,000 dilution). The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1 / 5,000 dilution) in TBST containing 5% nonfat dry milk for 1 hour at room temperature. The membranes were washed again with TBST, and signals were detected using chemiluminescence (Bio-Rad, Saint-Laurent, QC).

[0201] animal Female CD-1 nude mice (Crl:NU-Foxn1 nuFemale immunocompatible C57BL / 6 mice (C57BL / 6NCrl, 4-6 weeks old) were used for xenograft tumor models, and female immunocompatible C57BL / 6 mice (C57BL / 6NCrl, 4-6 weeks old) were used for allogeneic tumor models. All mice were obtained from Charles River Laboratories (St-Constant, QC). Animals were allowed to acclimate for 5 days before experiments. All mice were housed in a pathogen-free environment and handled in accordance with the Canadian Council on Animal Care (CCAC) guidelines for the care and use of laboratory animals.

[0202] Preparation of test articles for injection TH1902 (acetyl-GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY) was synthesized as previously described (PCT Publication No. WO 2017 / 088058). A 10 mg / ml stock solution of TH1902 was prepared in sterile aliquots and stored frozen. On the day of animal administration, the frozen aliquots were thawed and then diluted with sterile 5% dextrose injection USP (D5W) to the desired concentration for injection. A vehicle frozen stock solution was prepared identically to the dilution of the TH1902 stock solution, matching the amount of excipient present in the TH1902 animal groups. Docetaxel (Wonda Science Inc, Lexington, MA) was prepared on the same day as animal administration and matched the docetaxel content of the highest TH1902 dose administered for comparison. Docetaxel was dissolved in ethanol for injection to 50 mg / ml, then diluted to 25 mg / ml with polysorbate 80, and then diluted with sterile 5% D5W to the desired concentration for injection (i.e., 2.5 mg / ml). All diluted solutions were filtered (Millex-GP 0.22 μm syringe filter, PES membrane, Millipore) before animal administration. In terms of docetaxel content, a docetaxel dose of 15 mg / kg corresponds to 35 mg / kg of TH1902. Similarly, a docetaxel dose of 7.5 mg / kg corresponds to 17.5 mg / kg of TH1902 (the conversion factor 2.3 accounts for the molecular weight of the two docetaxel moieties within each TH1902 molecule). Therefore, docetaxel accounts for 44% of each TH1902 molecule. Anti-PD-L1 mAb (clone 10F.9G2; BP0101) and isotype control (clone LTF-2; BP0090) were purchased from Bio X Cell (Lebanon, NH) and diluted to the desired concentrations for injection according to the manufacturer's instructions (IP0065 and IP0070, respectively). All diluted solutions were filtered (Millex-GP 0.22 μm syringe filter, polyethersulfone (PES) membrane, MilliporeSigma, Burlington, MA) before animal administration.

[0203] In vivo therapeutic efficacy evaluation of docetaxel and TH1902 in the MDA-MB-231 immunodeficient chimeric model and the B16-F10 melanoma allogeneic immunocompetent model.

[0204] The MDA-MB-231 xenograft model has been previously described [4,5]. The B16-F10 melanoma syngeneic model was generated as follows: B16-F10 cells were resuspended in 100 μl of transplantation medium (MilliporeSigma, HBSS, #H6648) and Matrigel (Corning Inc., Corning, NY, #356231) at a 1:1 ratio, and 100 μl (1 × 10 6 cells / ml) in 1 × 10 5 Tumors were established in the dorsum of immunocompetent C57BL / 6 mice by subcutaneous inoculation of B16-F10 cells under light isoflurane anesthesia. The injection schedule, dose, number of treatment cycles, and method of administration of the injected substances are detailed in the figures. Specifically, mice were treated weekly with vehicle, docetaxel (15 mg / kg), or TH1902 (35 mg / kg) via intravenous (IV) tail vein injection. Treatments were administered until MDA-MB-231 tumors reached an average size of approximately 100 mm. 3 Treatment was initiated when the tumor reached 100 mg / kg / day or 3 days after implantation of B16-F10 cells. MDA-MB-231 tumors were harvested (i) on day 4 after three treatments with either vehicle, docetaxel, or TH1902; (ii) on day 4 after six treatments with TH1902; or (iii) on day 4 after six treatments with TH1902 followed by three cycles without treatment to observe long-term tumor growth. B16-F10 tumors were harvested on day 4 after two cycles of treatment with either vehicle, docetaxel, or TH1902. Tumors were harvested at each time point, fixed in 10% buffered formalin, and processed for immunohistochemistry (IHC) analysis. In all in vivo studies, tumor growth was monitored by two-dimensional measurements using electronic calipers, and tumor volume was calculated according to the following formula: tumor volume (mm ) = tumor volume (mm ) + ... 3 ) = π / 6 x length x width 2 Animal weights were measured with an accuracy of ±10 mg.

[0205] In vivo evaluation of the therapeutic efficacy of docetaxel or TH1902 in combination with an anti-PD-L1 antibody in the B16-F10 syngeneic model.

[0206] The B16-F10 melanoma syngeneic model was generated as described above. In the first study, the effects of docetaxel, TH1902, or anti-PD-L1 alone or in combination (docetaxel / anti-PD-L1 or TH1902 / anti-PD-L1) on primary tumor growth were investigated 3 days after B16-F10 cell implantation in mice. Treatment was administered in two cycles. The treatment dose and schedule for each cycle (shown in the figure legends) were as follows: The study included: (i) a control group treated with the appropriate vehicle and anti-PD-L1 isotype control (9 mg / kg, intraperitoneally, every other week); (ii) docetaxel at half the maximum tolerated dose (MTD, 7.5 mg / kg, intravenously, weekly); (iii) TH1902 at a dose equivalent to docetaxel (17.5 mg / kg, intravenously, weekly); (iv) a combination of docetaxel and anti-PD-L1; and (v) a combination of TH1902 and anti-PD-L1. The second study investigated the effect of increasing doses of TH1902 (4.37, 8.75, or 17.5 mg / kg, intravenously, weekly) or anti-PD-L1 (9 mg / kg, intraperitoneally, every other week), alone or in combination, on mouse survival. Treatment cycles continued until one of the study endpoints was reached (tumor size >2,000 mm). 3 , weight loss >20% of initial body weight, ulcerated tumor, death). In both studies, tumor growth and mouse weight were monitored as described above.

[0207] Evaluation of the in vivo therapeutic efficacy of docetaxel and TH1902 using a syngeneic Lewis lung carcinoma (LL / 2) xenograft model.

[0208] Tumors were grown at 1x10 in 100µl (LL / 2) HBSS / Matrigel (50:50). 6Cells were established by subcutaneous inoculation. All cells were injected into the dorsum of immunocompetent C57BL / 6 mice under mild isoflurane anesthesia. For LL / 2 tumors, mice were treated weekly for 11 days with vehicle, docetaxel (15 mg / kg), or TH1902 at an equivalent docetaxel dose (35 mg / kg) via intravenous (IV) tail vein injection starting on day 3 after implantation, including two treatments (days 0 and 7 after treatment initiation). In all studies shown, tumor growth was monitored by two-dimensional measurements performed using electronic calipers, and tumor volume was calculated according to the following formula: tumor volume (mm 3 ) = π / 6 x length x width 2 Animal weights were measured with an accuracy of ±10 mg. The vehicle group of mice was approximately 600–1000 mM 3 Tumors were harvested when they reached a size of 100 μg / ml.

[0209] Immunohistochemical staining of tissue microarrays and in vivo tumors. SORT1 expression was assessed at the Institute of Immunology and Cancer Research (IRIC, Montreal, QC) using high-density tissue microarrays (TMA) of human melanoma (IMH-366, Novus Biologicals, Centennial, CO) and healthy tissue (IMH-373, Novus Biologicals). TMAs were provided with subtype information, allowing for the evaluation of SORT1 expression in melanoma samples of different grades. Immunostaining of TMAs was performed as previously described [5]. An expert pathologist analyzed the IHC images. SORT1 labeling was assessed using the IHS method on a 0-3 scale: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Raw data were converted to an IHS score by multiplying the quantity and staining intensity scores. The resulting IHS score ranged from 0 to 12.

[0210] Primary MDA-MB-231 and B16-F10 tumors were harvested after euthanasia, fixed in 10% buffered formaldehyde, and embedded in paraffin. Hematoxylin and eosin (H&E; 23-314631 and 23-245657, Fisher Scientific) staining was performed to assess general morphology. IHC was performed on serial slides on a Leica Bond Max automated platform (Leica Biosystems, Nussloch GmbH) using a band polymer purification detection kit (DS9800, Leica Biosystems). Briefly, 4-micron sections from tissue blocks were removed onto coated slides. These were then deparaffinized, hydrated, and blocked with hydrogen peroxide. Heat-induced or enzymatic antigen retrieval was performed using buffers appropriate for each antibody. Slides were incubated with primary antibodies against SORT1, Ki-67, CD31, STING, CD45, CD3, CD8, CD4, FoxP3, CD161c, F4 / 80, CD68, CD206, perforin, granzyme B, or cleaved caspase-3, followed by secondary antibodies. Staining was completed with diaminobenzidine (DAB) chromogen, and hematoxylin was used as a control stain. Quantification of immune cells infiltrating the tumor nucleus was performed using National Institutes of Health (NIH) ImageJ version 1.4.21 software. The mean color intensity at identical threshold settings for each stain was divided by the surface area of ​​the tumor region of interest and expressed as a percentage of the positive area. For each section, analysis was performed on the entire tumor surface, excluding the outer edge, invasive margin, and necrotic areas. Details regarding all primary antibodies, commercial suppliers, dilutions, antigen retrieval, and processing conditions are listed in Table 7. [Table 7-1] [Table 7-2] 1 Primary antibody dilution with or without blocking solution (Bk; Leica Biosystems, No. PV6122). 2 H1: HIER 1, citrate buffer pH 6, Leica Biosystems, No. AR9961; HIER 2, EDTA buffer pH 9, Leica Biosystems, No. AR9640; ENZ: enzymatic digestion, Leica Biosystems, No. AR9551. 3 Incubation primary antibody / secondary antibody / polymer.

[0211] Detection of healthy blood vessels and VM-associated structures was performed as previously described [4]. Briefly, formalin-fixed, paraffin-embedded tumor tissue sections were stained with a primary antibody against mouse CD31. Next, sections were treated with 1% Schiff's persulfate (PAS) solution for 10 minutes. After a 2-minute wash with distilled water, tumor tissue sections were placed in Schiff's solution for 30 minutes in a dark room and then washed three times with distilled water. Finally, slides were counterstained with Leica's proprietary hematoxylin and mounted for analysis. Healthy blood vessels are CD31-positive / PAS-positive, whereas VM-associated structures are CD31-negative / PAS-positive. Images were acquired using a NanoZoomer slide scanner (Hamamatsu Photonics KK, Hamamatsu, Japan) and analyzed with an Aperio ImageScope (Leica Biosystems, version 12.4.3.5008, Buffalo Grove, IL).

[0212] Cell proliferation assay. To evaluate the effects of docetaxel and TH1902 on the proliferation of SK-MEL-28 and B16-F10 melanoma cells, cells were seeded into 96-well plates (PerkinElmer, Waltham, MA) and treated with various concentrations of the drugs in complete cell culture medium. After 120 h of culture for SK-MEL-28 and 72 h for B16-F10, cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the protocol described by Mosmann

[21] with the following modifications: Cells were incubated with MTT (0.5 mg / ml) for 4 h at 37°C in a humidified atmosphere containing 5% CO2. After incubation, 100 μL of DMSO (lysis reagent) was added to each well and mixed thoroughly for 5 min to dissolve the dark blue crystals. The presence of viable cells was visualized by the development of a purple color due to the formation of formazan crystals. Plates were read on a SpectraMax™ Plus reader (Molecular Devices, San Jose, CA) using a test wavelength of 570 nm and a reference wavelength of 650 nm. Each condition was analyzed in quadruplicate.

[0213] Cell apoptosis assay. Annexin V / PI staining was performed using an apoptosis detection kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. Briefly, B16-F10 cells were treated with serum-free medium containing docetaxel (0.1 or 2 μM) or TH1902 (0.05 or 1 μM) for 15 minutes. Cells were then washed twice with complete growth medium and incubated for 94 hours in complete growth medium. Cells were harvested and resuspended in 100 μL of 1X binding buffer containing 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI). Cells were finally incubated for 15 minutes at room temperature in the dark, and the number of apoptotic cells was acquired and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ).

[0214] Senescence activity assay. B16-F10 cells were seeded at a density of 20,000 cells per well in ibidi (Grafelfing, Germany) 8-well chamber slides. After 24 hours, cells were treated with serum-free medium containing 7.5 μM docetaxel or 3.75 μM TH1902 for 2 hours. Cells were then washed twice with complete growth medium and incubated in complete growth medium for 4 days. As a positive control for senescence, cells were treated with 12.5 μM etoposide in serum-free medium for 24 hours, washed, and then incubated in complete growth medium for up to 4 days. Senescence-associated β-galactosidase (SA-β-gal) activity was detected using the CellEvent™ Senescence Green Detection Kit (Thermo Fisher, #C10850) according to the manufacturer's instructions. Photomicrographs were taken at 20x magnification using a confocal microscope (Nikon A1plus, Melville, NY) and digitized, then analyzed using NIH ImageJ version 1.4.21 software. For cell morphology assessment, cells were fixed in 10% formalin phosphate, stained with 0.1% crystal violet / 20% methanol, and observed under a microscope.

[0215] Total RNA isolation, cDNA synthesis, and real-time quantitative PCR. Total RNA was extracted from cell monolayers using the Qiagen RNeasy™ kit (QIAGEN, Toronto, ON). For cDNA synthesis, 2 μg of total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems, Foster City, CA). cDNA was stored at -20°C prior to polymerase chain reaction (PCR). Gene expression was quantified by real-time quantitative PCR using SsoFast EvaGreen Supermix™ (1725201, Bio-Rad, Hercules, CA). DNA amplification was performed using an Icycler iQ5 (Bio-Rad), and product detection was performed by measuring the binding of the fluorescent dye SYBR Green I to double-stranded DNA. The following primer sets were from QIAGEN: interleukin-6 (IL-6) (QT00098875, Mm_Il6_1_SG), tumor necrosis factor alpha (TNFα) (QT00104006, Mm_Tnf_1_SG), GAPDH (QT01658692, Mm_Gapdh_3_SG), and peptidylprolyl isomerase A (PPIA) (QT00247709, Mm_Ppia_1_SG). The relative amounts of target gene mRNA were normalized to the internal housekeeping genes PPIA and GAPDH. RNA was analyzed using an amplification plot (fluorescence signal vs. cycle number) to determine ΔC. T The difference between the mean values ​​of triplicate samples of the target gene and the housekeeping gene (ΔC T ) was calculated using CFX Manager Software version 2.1 (Bio-Rad), and the relative quantification value (RQV) was calculated as 2 -ΔCT was expressed as:

[0216] Flow cytometry. Single live B16-F10 cell suspensions were washed with RPMI-1640 supplemented with 8% FBS in PBS and then washed once with FACS buffer. The samples were then incubated with Fc-blocking mouse (2%) and mouse serum (5%) for 10 minutes, then stained with anti-MHC-I or anti-PD-L1 antibodies for 1 hour at 4°C in the dark, and finally washed twice with PBS containing 0.5% BSA. Samples were acquired using an LSR Fortessa (BD Biosciences), and the results were analyzed using FlowJo software.

[0217] statistical analysis Data are expressed as mean ± standard error of the mean (SEM) or standard deviation (SD), as indicated in the figure legends. Statistical analysis was performed using a t-test to compare two samples, and for three or more samples, one-way analysis of variance with Bonferroni, Dunnett, or Tieki multiple comparisons was used. Values ​​of p<0.05 (*) and p<0.01 (**) were considered significant, and asterisks (*) indicate such levels of significance in the figures.

[0218] Example 2: Sustained and long-term antitumor activity of TH1902 in an immunosuppressed MDA-MB-231 TNBC-derived xenograft model correlates with increased immune cell infiltration and STING expression.

[0219] The efficacy of TH1902 and docetaxel in a TNBC xenograft model was first investigated in vivo. Immunosuppressed hairless mice were implanted with MDA-MB-231 cancer cells in the right flank, and tumor growth was monitored as described in Example 1. Mice were treated with an IV bolus injection of either (i) 15 mg / kg / week docetaxel at the maximum tolerated dose (MTD) (three treatments) or (ii) 35 mg / kg / week TH1902 (six treatments). An additional group (iii) received TH1902 for six treatments and was observed until day 60 (Figure 1A). Treated tumors were harvested after the end of treatment, or on day 60 in the case of TH1902. In contrast to docetaxel, comparable TH1902 doses only halved tumor growth, whereas TH1902 induced complete tumor regression, an effect that was maintained throughout the six treatments and continued until day 60 (i.e., 25 days after the last treatment). Body weight remained largely unchanged (Figure 1E). H&E staining of excised tumors revealed a significant expansion of SORT1-positive cancer cell morphology after six treatments, and this morphology persisted even after treatment cessation (Figure 1B). IHC analysis further demonstrated decreased Ki67 cell proliferation and increased STING and CD45 staining in TH1902-treated tumors, demonstrating persistent SORT1 expression in cancer cells after treatment (Figure 1C). Increased CD45 immune infiltrate staining after treatment particularly surrounded the expanded cancer cells (Figure 1C). The anti-vascular structure (VM) properties of TH1902 were observed from the third cycle of treatment, with CD31 - / PAS +Staining was reduced in TH1902-treated tumors (Figure 1D). In contrast, docetaxel was much less effective than TH1902 with respect to Ki67, STING, CD45 staining, and vascular structures (VM). Interestingly, unlike docetaxel, TH1902 significantly induced senescence-associated expression of p21 and p53 in MDA-MB-231 cells in vivo (Figure 1F–G). Finally, the results shown in Figure 1H–I demonstrate that treatment with TH1902 increases PD-L1 expression in MDA-MB-231 tumors in vivo. Collectively, these results provide evidence that TH1902 has the ability to alter the immune-tumor microenvironment (iTME) through immune cell infiltration processes, which correlates with a durable effect on tumor regression.

[0220] Example 3: SORT1 shows high expression in clinically annotated melanoma tissues and cell line models.

[0221] To evaluate whether TH1902 could function as a SORT1 receptor-mediated chemotherapy approach in an immunocompatible syngeneic model, SORT1 expression was assessed in murine B16-F10 melanoma cells and other cell lines. This screening was also performed on TMAs of stage II-IV melanomas clinically annotated by IHC as healthy skin tissue (Figure 2A). IHS scoring showed that SORT1 expression increased across these stages, whereas levels remained low in healthy tissue (Figure 2B). High SORT1 expression was also verified in lysates from the murine B16-F10 melanoma cell line and two human melanoma cell lines (A375 and SK-MEL-28), as well as a positive control (human TNBC-derived MDA-MB-231) (Figure 2C). High SORT1 expression in murine B16-F10 cells validated that such a syngeneic model could be used to evaluate the in vivo therapeutic effects of TH1902 on the iTME.

[0222] Example 4: TH1902 exhibits antiproliferative and inducing apoptosis activity in vivo and causes senescence in cancer cells.

[0223] SORT1-positive SK-MEL-28 (Figure 3A) and B16-F10 (Figure 3B) melanoma cells were selected to test the antiproliferative effects of docetaxel and TH1902. The biological effects of TH1902 were monitored, and the half-maximal inhibitory concentration (IC) of TH1902 was determined. 50 ) were similar to those of docetaxel, 0.38 nM vs. 0.39 nM in human SK-MEL-28 cells and 2.57 nM vs. 1.72 nM in murine B16-F10 cells, respectively (Figure 3C), indicating that the antiproliferative properties of docetaxel are unaffected by the conjugation of the cleavable linker to TH19P01. Because docetaxel's cytotoxicity is related to its ability to induce cell cycle arrest and apoptosis, we investigated the apoptotic effects of low and high concentrations of docetaxel and TH1902 on B16-F10 cells. Cells were treated with 0.1 μM or 2 μM docetaxel (Figure 3D, black bars) or 0.05 μM and 1 μM TH1902 (with equimolar bound docetaxel concentrations, Figure 3D, gray bars) for 15 min, washed, and then further incubated in complete medium for up to 96 h. Apoptosis was then assessed by flow cytometry as described in Example 1. While increasing concentrations of docetaxel induced apoptosis, TH1902 induced a dose-dependent, stepwise increase in apoptosis (Figure 3D). Because docetaxel is known to induce senescence in B16-F10 cells

[22] and limit their proliferative capacity

[23] , the respective effects of docetaxel and TH1902 on senescence were then tested as described in Example 1 (Figure 3E). Compared to control conditions, SA-β-gal activity-dependent senescence markers were increased in docetaxel-treated cells and cells treated with the senescence inducer etoposide (positive control). Compared to docetaxel, TH1902 further increased senescence measured by SA-β-gal activity (Figure 3F). The increased senescence induction was further confirmed morphologically in TH1902-treated cells, which largely flattened out compared to control or docetaxel conditions (Figure 3G). This data indicates that the effects of increased apoptosis and senescence may support the antitumor effect of TH1902 on B16-F10 melanoma cells.

[0224] Example 5: TH1902 inhibits tumor growth and induces leukocyte infiltration in an immunocompetent syngeneic "cold" tumor model.

[0225] B16-F10 melanoma syngeneic tumors were generated (Figure 4A), and tumor size was monitored as described in Example 1. Tumors in vehicle-treated xenograft-bearing mice grew at an exponential rate (Figure 4A, filled circles). Partial inhibition of tumor growth was observed after intravenous administration of 15 mg / kg / week of docetaxel (Figure 4A, squares), whereas treatment with an equivalent dose of TH1902 (35 mg / kg / week) caused tumor shrinkage after two treatments within the measured period (Figure 4A, triangles). Due to rapid tumor growth, test article administration was limited to two doses on a weekly schedule. B16-F10 melanoma tumors from mice treated with vehicle, docetaxel, or TH1902 were then excised, fixed in formalin, and examined immunohistochemically (Figure 4B). Within the tumor shown, there are areas of necrosis, as previously observed within TH1902-treated MDA-MB-231 tumors (Figure 4C), with some areas filled with actual cancer cells with significantly enlarged morphology. Again, significant infiltration of immune cells (CD45+ total leukocytes) into tumors from TH1902-treated animals was detected (Figure 4D). The tumor stroma of vehicle-treated animals showed little CD45 immunostaining (Figure 4C, left panel), indicating the absence of leukocytes, while tumors from docetaxel-treated animals showed slightly higher leukocyte infiltration (Figure 4C, middle panel). However, in both cases, staining was limited to the outer periphery of the tumor. This indicates that the tumors are of an immune-excluded phenotype, rather than the related immune-desert phenotype, in which T cells are significantly absent from the tumor parenchyma or stroma

[24] . It is well established that tumors lacking lymphocytic infiltration are unlikely to respond to checkpoint inhibitors (CPIs), a finding well established for lymphocytic infiltration [25, 26]. However, animals treated with TH1902 exhibited significantly higher tumor levels of staining for the pan-immune cell marker CD45 (Figure 4C, right panel), and quantification confirmed the infiltration of most leukocytes within the tumor parenchyma (Figure 4D). Body weights of mice treated with test article or vehicle were tracked as a crude indicator of morbidity.B16-F10 tumor-bearing mice had similar body weights whether administered vehicle, docetaxel, or TH1902 (FIG. 4E), indicating that the animals' weights remained within an acceptable range.

[0226] Example 6: TH1902 induces immune cell infiltration within tumors.

[0227] The immune response to tumors is a complex orchestration involving numerous cell types, interacting membrane proteins, and soluble effectors

[27] . Docetaxel moderately induced lymphocyte infiltration of all subclasses, including cytotoxic T cells, helper T cells, regulatory T cells, and innate immune cells (NK cells), within the tumor parenchyma by IHC (Figure 5A, horizontal center panel). However, the increase with TH1902 treatment was systematically and significantly greater than that with docetaxel in all measured lymphocyte classes (Figure 5A, lower horizontal panel, and Figure 5B, gray bars). The CD3 T cell marker was slightly increased with docetaxel and significantly increased with TH1902 compared to vehicle. IHC data also showed that TH1902 treatment significantly increased the expression of CD8+ (or cytotoxic) T cells, known to be involved in tumor immune responses. Furthermore, TH1902 increased CD4 T cell infiltration more than docetaxel, while TH1902 induced increased levels of infiltrating regulatory T cells (Treg, FoxP3).Furthermore, expression of the CD161c NK marker was increased in TH1902-treated mice compared with vehicle- or docetaxel-treated mice (Figure 5B).

[0228] Macrophages also represent an important class of immune cells within tumors. Indeed, tumor-associated macrophages (TAMs) are generally the most abundant immune cell population within most tumors

[28] . TAMs can enhance or antagonize the cytotoxic activity of immune cells, and these effects are often attributed to two distinct subpopulations, M1 and M2, respectively

[29] . These are not two distinct macrophage lineages, but rather cells whose "polarity" is switched to M1 or M2 by local humoral factors

[30] . Similar to tumor-infiltrating lymphocytes (TILs), tumor-infiltrating TAMs (F4 / 80 + ), and M1 (CD68 + ) and M2(CD206 + The number of macrophages was slightly increased by docetaxel treatment but significantly increased by TH1902 treatment (Figures 5C and 5D). + Expression of M2 macrophage markers was significantly higher in TH1902 than in CD68 + The same trend was observed for M1 macrophages. Thus, the stimulation of leukocyte infiltration into tumors by TH1902 applies to multiple immune cell classes.

[0229] Example 7: TH1902 induces immune-stimulated apoptosis.

[0230] Regardless of the specific lineage of cells infiltrating tumors, the efficacy of treatment often depends on the induction of apoptosis in tumor cells

[31] . One mechanism by which immune cells (primarily NK cells and cytotoxic T cells) eliminate other cells is via the granzyme B / perforin apoptosis pathway. Therefore, the effects of both docetaxel and TH1902 on this pathway were investigated. When animals were treated with docetaxel, there was a small increase in perforin and granzyme B, but no increase in caspase-3, compared to the vehicle-treated group (Figure 6A). However, the significant (5- to 7-fold) increase in perforin, granzyme B, and caspase-3 staining after treatment with TH1902 indicates that TH1902-induced tumor cell apoptosis is mediated, at least in part, by immune cells (Figure 6B). Overall, these data suggest that CD8 + Supporting the significant effect of TH1902 on T cell effector and NK cell anti-tumor responses, this leads to complete tumor regression after cancer cell death.

[0231] Example 8: Effect of TH1902 in combination with anti-PD-L1 on B16-F10 tumors.

[0232] Treatment of B16-F10 tumors with TH1902 appears to broadly allow immune cell infiltration into tumors and greatly enhance immune cell-mediated apoptosis. TH1902 also appears to circumvent the immune-exclusionary properties of these "cold" tumors. Consequently, the combination of TH1902 with checkpoint inhibitors (CPIs) appears feasible. Because anti-PD-L1 immunotherapy is known to be ineffective in the B16-F10 allogeneic model

[32] , treatment with this antibody in combination with docetaxel or TH1902 was evaluated. To assess synergy, the doses of docetaxel and TH1902 were halved. As observed, tumor growth was similar in mice treated with vehicle and isotype control antibody (anti-PD-L1 control), whereas administration of anti-PD-L1 alone reduced tumor growth by 35%, although the reduction was small and not statistically significant (p=0.0702). These results are similar to previous reports in this tumor model

[33] , where docetaxel administration resulted in a small but significant reduction of tumor growth by 49%, while TH1902 resulted in a much larger and significant reduction of tumor growth by 92% (Figure 7A). While anti-PD-L1 therapy combined with docetaxel only reduced tumor growth by 66%, the combination of anti-PD-L1 and TH1902 significantly reduced tumor size, demonstrating tumor shrinkage by 11 days (30% reduction compared to the initial tumor volume at the start of treatment). The enhanced tumor growth suppression achieved by the combination of TH1902 and anti-PD-L1 became more apparent as the experiment progressed. While single-agent administration of docetaxel or anti-PD-L1 only partially suppressed tumor growth, TH1902 effectively reduced this growth after two cycles (Figure 7A). Interestingly, while the anti-PD-L1 / docetaxel combination failed to further reduce tumor growth, the anti-PD-L1 / TH1902 combination showed a significant and greater reduction in tumor growth at day 14 post-treatment compared to that obtained with TH1902 alone. Both docetaxel and TH1902 appeared to be well tolerated, with mouse body weights being largely unaffected compared to the control group (Figure 7D).However, anti-PD-L1 used alone or in combination with docetaxel or TH1902 caused similar weight loss, suggesting that the addition of TH1902 had limited effect on mouse weight (Figure 7D). Overall, these data support the therapeutic efficacy of TH1902 as monotherapy, which is significantly increased when combined with anti-PD-L1. We also demonstrate that TH1902 reverses cancer resistance to CPIs.

[0233] These results were unexpected because B16-F10 melanoma tumors are generally considered immunologically cold, with limited immune cell infiltration and poor response to immune checkpoint inhibitors (see https: / / drugdevelopment.labcorp.com / industry-solutions / oncology / preclinical / tumor-spotlights / b16-f10-a-murine-melanoma-model.html and Ueha et al., Cancer Immunol Res (2015) 3(6):631-640 [Figure 8]).

[0234] Example 9: Effect of different levels of TH1902 in combination with anti-PD-L1 on tumor growth in a survival study.

[0235] Next, a survival study was performed on mice treated with (or without) anti-PD-L1 along with different doses of TH1902. The results, shown in Figure 7C, indicate that TH1902 exhibited strong dose-dependent inhibition of tumor growth. Treatment with the lowest dose of TH1902 or anti-PD-L1 alone resulted in a small but non-significant tumor growth inhibition (TGI) compared to vehicle-treated animals (p=0.1394 and 0.1787, respectively), and 10 days after the start of treatment, tumor size was reduced by half, similar to vehicle-treated animals, with a median survival of 14 days (TH1902 4.37 mg / kg / week and anti-PD-L1, both p=0.0521) or 11.5 days (vehicle). However, tumors in animals treated with either of the higher doses of TH1902 were significantly smaller, demonstrating a 95% tumor growth inhibition (TGI) of the initial tumor volume at the start of treatment (8.75 mg / kg / week) or a 54% reduction (17.5 mg / kg / week) with median survival times of 17 and 24 days, respectively. Mice in the survival study tolerated anti-PD-L1 much better than those seen in the efficacy study. While no mice in this study showed significant weight loss, mice that did not receive IP injections of control IgG or anti-PD-L1 mAb showed greater weight gain, which may be due to the rapid exponential growth of some tumors. Survival curves were generated for each of the nine mouse groups. The two groups receiving 17.5 mg / kg / week TH1902 (with or without anti-PD-L1) showed the longest median survival times, 24 and 32.5 days, respectively (Figure 7B). Statistically, animals treated with high concentrations of TH1902 (8.75 and 17.5 mg / kg / week) as monotherapy or all three TH1902 doses (4.37, 8.75, and 17.5 mg / kg / week) in combination with anti-PD-L1 had significantly different survival curves compared to vehicle-treated animals (Table 8). Furthermore, the survival curves of these three combined TH1902 treatment groups were significantly different (p<0.05) from those of the TH1902-only treatment group, with increases in lifespan of 4.5 vs. 2.5 days, 10 vs. 5.5 days, and 21 vs. 12.5 days compared to vehicle, respectively (Table 8).The combination [TH1902 17.5 mg / kg / week / anti-PD-L1] significantly and synergistically increased animal survival compared to either anti-PD-L1 or TH1902 17.5 mg / kg / week as single agents (median survival increased by 21 days vs. anti-PD-L1 alone and 12.5 days vs. TH1902 17.5 mg / kg / week alone). [Table 8]

[0236] Example 10: TH1902 induces downstream effectors of the STING pathway and increases cell surface PD-L1 and MHC-I expression in B16-F10 melanoma cells.

[0237] Cellular senescence, an irreversible cell cycle arrest, represents a major barrier to tumorigenesis

[34] . Docetaxel has been reported to induce senescence in mouse lung and prostate tumor cells through a sustained DNA damage response

[37] [35,36]. Recently, DNA-damaging conditions have been shown to activate the cGAS (cyclic GMP-AMP synthase) / STING (stimulator of interferon genes) pathway in TNBC cells, a process associated with cell proliferation and invasiveness [38,39]. Given the ability of TH1902 to induce senescence in B16-F10 melanoma cells and suppress cell proliferation in melanoma (as shown herein) and TNBC cells, the involvement of the STING pathway was next investigated. Cell lysates were harvested after treatment with docetaxel or TH1902, and STING protein expression was assessed by immunoblotting (Figure 9A). While docetaxel induced STING expression by densitometry, TH1902 did so more effectively at concentrations up to 500 nM (Figure 9B). Evaluation of downstream effectors of the STING pathway (Figure 9C) revealed that TH1902 induced the expression of the p53 transcription factor, which is known to activate the cGAS / STING pathway. TH1902 also induced the expression of p21, which is known to maintain the survival of DNA damage-induced senescent cells (Figure 9D, gray bars). Consistent with activation of the STING pathway, we also observed a greater increase in the phosphorylation status of TBK1 and p65 with TH1902 treatment compared to docetaxel (Figure 9D). TBK1 is a key serine / threonine protein kinase that mediates NF-κB signaling and regulates the production of inflammatory cytokines and the activation of innate immunity

[43] . TBK1 mediates the phosphorylation and nuclear translocation of IRF3 independently of NF-κB, which contributes to the induction of type I interferon

[44] .

[0238] Next, cytokine modulation was assessed in docetaxel- and TH1902-treated cells. STING pathway activation further correlated with increased transcription levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF)α by TH1902 (Figure 9E). Finally, PD-L1 (Figure 9F, bottom panel) and MHC-I (Figure 9F, top panel) were also induced on the cell surface of TH1902-treated B16-F10 melanoma cells, all of which were also induced by interferon-gamma (Figure 9F, right panel). NF-κB-mediated MHC-I upregulation is thought to enhance T cell activation

[45] , which increases tumor cell recognition and ultimately converts melanoma cells into cytotoxic CD8 T cells. + These data suggest that increased NF-κB expression via p65 phosphorylation is associated with a favorable response to CPI therapy in melanoma patients [46,47]. TH1902 may enhance antitumor immunity, highlighting in vivo molecular evidence suggesting that compensatory upregulation of the CPI ligand PD-L1 may benefit from anti-PD-L1 counteraction and achieve maximal antitumor immunity in vivo, consistent with the synergistic effects achieved by the combination of TH1902 and anti-PD-L1 in Example 9.

[0239] Example 11: TNFα, but not IL-6, induces cell surface expression of PD-L1 and MHC-I in B16-F10 melanoma cells.

[0240] To decipher the potential crosstalk between TH1902-induced TNFα / IL-6 induction and increased levels of MHC-I and PD-L1, B16-F10 melanoma cells were treated with the indicated concentrations of TNFα or IL-6 for 96 hours. TNFα treatment was observed to increase the cell surface expression of PD-L1 and MHC-I in a dose-dependent manner (Figure 10A), whereas IL-6 treatment did not alter the cell surface expression of both proteins (Figure 10B). These data suggest that TNFα, rather than IL-6, is the primary trigger for TH1902-mediated induction of PD-L1 and MHC-I cell surface expression.

[0241] Example 12: Effect of TH1902 in an immunologically cold lung cancer model.

[0242] The effect of TH1902 on tumor growth was evaluated in a syngeneic Lewis lung carcinoma (LL / 2) model, another immunologically cold tumor model that expresses SORT1. LL / 2 tumor cells are resistant to immune checkpoint inhibitors (https: / / drugdevelopment.labcorp.com / industry-solutions / oncology / preclinical / tumor-spotlights / ll-2-an-immunosuppressive-murine-tumor-model.html, Figures 11A and 11B). The results, shown in Figure 11C, demonstrate that treatment with TH1902 results in almost complete suppression of LL / 2 tumor growth.

[0243] The results presented herein provide evidence that conjugates of sortilin-targeting peptides and chemotherapeutic drugs can induce immune cell infiltration and cytotoxic cell-mediated tumor killing in immunologically cold tumors, and that combining such conjugates with immune checkpoint inhibitors leads to tumor eradication, including tumors that are resistant to immune checkpoint inhibitor monotherapy.

[0244] Although the present invention has been described with reference to specific embodiments above, modifications can be made without departing from the spirit and essence of the invention as defined in the appended claims. In the claims, the term "comprising" is used as an open-ended term and is substantially equivalent to the phrase "including, but not limited to." The singular forms "a," "an," and "the" also include the corresponding plural forms unless the context clearly dictates otherwise.

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Claims

1. 1. A method for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy, comprising administering to the subject an effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound has the formula A-(B): ｎ wherein A is a peptide compound of 30 residues or less, which comprises an amino acid sequence having at least 60% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 13; X １ X ２ X ３ X ４ X ５ GVX ６ AKAGVX ７ NX ８ FKSESY (SEQ ID NO: 1) (X ９ ) ｎ GVX １０ AKAGVX １１ NX １２ FKSESY (SEQ ID NO: 2) YKX １３ LRRX １４ APRWDX １５ PLRD PALRX １６ X １７ L (SEQ ID NO: 3) YKX １８ LRR (X １９ ) Ｎ PLRD PALRX ２０ X ２１ L (SEQ ID NO: 4) IKLSGGVQAKAGVINMDKSESM (SEQ ID NO: 5) IKLSGGVQAKAGVINMFKSESY (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESYK (SEQ ID NO: 7) GVQAKAGVINMFKSESY (SEQ ID NO: 8) GVRAKAGVRNMFKSESY (SEQ ID NO: 9) GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 10) YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 11) YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 13) X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ , X １８ , and X １９ are independently selected from any amino acid, X １６ , X １７ , X ２０ , and X ２１ is independently selected from Q, P, Y, I, and L; n is 0, 1, 2, 3, 4 or 5 X ９ When X occurs multiple times, each X ９ are independently selected from any amino acid, X １９ When X occurs multiple times, each X ９ are independently selected from any amino acid, Optionally, the peptide compound is cyclic; The method, wherein B is at least one therapeutic agent, and B is attached to A directly or via a linker.

2. The method of claim 1, wherein the peptide compound comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1-13.

3. 3. The method of claim 1 or 2, wherein the peptide compound comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 13 and further comprises 1 to 3 additional amino acids at the amino and / or carboxy termini thereof.

4. The method of claim 3 , wherein the peptide compound comprises a cysteine ​​residue at its amino and / or carboxy terminus.

5. The peptide compound comprises one of the following amino acid sequences: Z １ X １ X ２ X ３ X ４ X ５ GVX ６ AKAGVX ７ NX ８ FKSESYZ ２ (SEQ ID NO: 34) Z １ (X ９ ) ｎ GVX １０ AKAGVX １１ NX １２ FKSESYZ ２ (SEQ ID NO: 35) Z １ YkX １３ LRRX １４ APRWDX １５ PLRD PALRX １６ X １７ LZ ２ (SEQ ID NO: 36) Z １ YKX １８ LRR(X) １９ ) Ｎ PLRDPALRX ２０ X ２１ LZ ２ (Allocation number 37) Z １ IKLSGGVQAKAGVINMDKSESMZ ２ (SEQ ID NO: 38) Z １ IKLSGGVQAKAGVINMFKSESYZ ２ (SEQ ID NO: 39) Z １ IKLSGGVQAKAGVINMFKSESYKZ ２ (SEQ ID NO: 40) Z １ GVQAKAGVINMFKSESYZ ２ (SEQ ID NO: 41) Z １ GVRAKAGVRNMKSESYZ ２ (SEQ ID NO: 42) Z １ GVRAKAGVRN(Nle)FKSESYZ ２ (SEQ ID NO: 43) Z １ YKSLRRKAPRWDAPLRDPALRQLLZ ２ (SEQ ID NO: 44) Z １ YKSLRRKAPRWDAYLRDPALRQLLZ ２ (SEQ ID NO: 45) Z １ YKSLRRKAPRWDAYLRDPALRPLL Z ２ (SEQ ID NO: 46), X １ ~X ２１ is as defined in claim 1, and Z １ is a cysteine ​​residue or is absent, Z ２ is a cysteine ​​residue or is absent, Z １ and Z ２ The method of claim 4 , wherein at least one of

6. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO: 1 or 2.

7. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO: 3 or 4.

8. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

5.

9. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

6.

10. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

7.

11. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

8.

12. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

9.

13. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

10.

14. 14. The method of claim 13, wherein the peptide compound comprises the amino acid sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 47).

15. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

11.

16. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

12.

17. The method of claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

13.

18. The method of any one of claims 1 to 17, wherein the peptide compound comprises at least one modifying group at its amino and / or carboxy terminus.

19. 18. The method of claim 17, wherein the at least one modifying group is acetyl or succinyl.

20. 2. The method of claim 1 , wherein the peptide compound is represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:

47. Acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14) Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15) Acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16) Acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17) Acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18) Acetyl-GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 48).

21. 21. The method of any one of claims 1 to 20, wherein B is attached to A at a free amine of the peptide compound, an N-terminal position of the peptide compound, a free -SH of the peptide compound, and / or a free carboxyl of the peptide compound.

22. 22. The method of any one of claims 1 to 21, wherein B is linked to A via a linker.

23. The conjugate is represented by SEQ ID NO: 23 or SEQ ID NO: 24: GVRAK (J) １ ) AGVRN (Nle) FK (J ２ ) SESY (array number 22), Acetyl-GVRAK(J １ ) AGVRN (Nle) FK (J ２ ) SESY (array number 23), Here, J １ and J ２ and each independently is a therapeutic agent attached to a lysine (K) residue.

24. The method of any one of claims 1 to 23, wherein the therapeutic agent is an anti-tumor agent, such as a radionuclide or a chemotherapeutic agent.

25. 25. The method of claim 24, wherein the chemotherapeutic agent is a taxane.

26. 26. The method of claim 25, wherein the chemotherapeutic agent is docetaxel.

27. The method of any one of claims 1 to 26, further comprising treating the subject with immunotherapy.

28. 28. The method of any one of claims 1 to 27, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

29. 29. The method of claim 28, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

30. 30. The method of claim 28 or 29, wherein the ICI is a blocking antibody.

31. The method of claim 28 or 29, wherein the ICI is a PD-L1 inhibitor.

32. The method of any one of claims 1 to 31, wherein the cancer is an immunologically cold cancer.

33. 33. The method of any one of claims 1 to 32, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

34. 27. Use of a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

35. 27. Use of a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer, and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

36. 36. The use of claim 34 or 35, wherein the conjugate compound, a pharmaceutically acceptable salt thereof, or a medicament is for use in combination with immunotherapy.

37. The use according to any one of claims 34 to 36, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

38. 38. The use of claim 37, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

39. 39. The use according to claim 37 or 38, wherein the ICI is a blocking antibody.

40. The use according to claim 38 or 39, wherein the ICI is a PD-L1 inhibitor.

41. The use according to any one of claims 34 to 40, wherein the cancer is an immunologically cold cancer.

42. The use according to any one of claims 34 to 41, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

43. 27. A conjugate compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 26 for use in (i) enhancing an anti-tumor immune response in a subject suffering from a sortilin-expressing cancer and / or (ii) treating a subject suffering from a sortilin-expressing cancer that is resistant to immunotherapy.

44. 44. A conjugate compound or a pharmaceutically acceptable salt thereof for use in accordance with claim 43, wherein the peptide compound is for use in combination with immunotherapy.

45. 45. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to claim 43 or 44, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

46. 46. ​​The conjugate compound or a pharmaceutically acceptable salt thereof for use according to claim 45, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

47. 47. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to claim 45 or 46, wherein said ICI is a blocking antibody.

48. 48. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to claim 46 or 47, wherein said ICI is a PD-L1 inhibitor.

49. 49. A conjugate compound or a pharmaceutically acceptable salt thereof for use according to any one of claims 43 to 48, wherein the cancer is an immunologically cold cancer.

50. 50. The conjugate compound or a pharmaceutically acceptable salt thereof for use according to any one of claims 43 to 49, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

51. 27. A method for treating a sortilin-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, in combination with immunotherapy.

52. 52. The method of claim 51, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

53. 53. The method of claim 52, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

54. 54. The method of claim 52 or 53, wherein the ICI is a blocking antibody.

55. The method of claim 53 or 54, wherein the ICI is a PD-L1 inhibitor.

56. 56. The method of any one of claims 51 to 55, wherein the cancer is an immunologically cold cancer.

57. 57. The method of any one of claims 51 to 56, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

58. 58. The method of any one of claims 51 to 57, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are present in different compositions.

59. 58. The method of any one of claims 51 to 57, wherein the conjugate compound or pharmaceutically acceptable salt thereof and the immunotherapy are present in the same composition.

60. 27. Use of a conjugate compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 26 in combination with immunotherapy for the treatment of sortilin-expressing cancer.

61. 27. Use of a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, in combination with immunotherapy, for the manufacture of a medicament for the treatment of sortilin-expressing cancer.

62. 62. The use of claim 60 or 61, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

63. 63. The use of claim 62, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

64. 64. The use of claim 62 or 63, wherein the ICI is a blocking antibody.

65. The use according to claim 63 or 64, wherein the ICI is a PD-L1 inhibitor.

66. The use according to any one of claims 60 to 65, wherein the cancer is an immunologically cold cancer.

67. 67. The use according to any one of claims 61 to 66, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

68. 68. The use of any one of claims 61 to 67, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are present in different compositions.

69. 68. The use of any one of claims 61 to 67, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are present in the same composition.

70. 27. A combination therapy for use in the treatment of sortilin-expressing cancer, comprising a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and immunotherapy.

71. 71. The combination therapy for use according to claim 70, wherein said immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

72. 72. The combination therapy for use according to claim 71, wherein the ICI is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

73. 73. A combination therapy for use according to claim 71 or 72, wherein the ICI is a blocking antibody.

74. 74. The combination therapy for use according to claim 72 or 73, wherein the ICI is a PD-L1 inhibitor.

75. 75. A combination therapy for use according to any one of claims 71 to 74, wherein said cancer is an immunologically cold cancer.

76. 76. The combination therapy for use according to any one of claims 70 to 75, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

77. 77. A combination therapy for use according to any one of claims 70 to 76, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are present in different compositions.

78. 77. A combination therapy for use according to any one of claims 70 to 76, wherein said conjugate compound or a pharmaceutically acceptable salt thereof and said immunotherapy are present in the same composition.

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