Drug conjugate compounds for stimulating anti-tumor immune responses

Abstract

Despite the development of an immune checkpoint inhibitor (ICI), cancer treatment has gained significant progress, but several cancers still exhibit resistance to these agents. Certain tumors are considered to be immune cold tumors, which are typically poorly responsive to cancer immunotherapies such as immune checkpoint blocking (ICB) therapies, and thus there is a need to develop new methods for treating such cancers. Disclosed herein are methods for enhancing an anti-tumor immune response and / or treating a subject suffering from a sortin-expressing cancer by administering a conjugate compound comprising a sortin targeting agent conjugated to an anti-cancer agent, methods and uses in subjects suffering from cancer, such as cancer that is resistant to immunotherapy. Combination therapies comprising the conjugate compounds and an immunotherapy such as ICB are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,561, filed on November 14, 2022. The entire contents of this application are incorporated herein by reference.

[0003] Sequence Listing

[0004] The Sequence Listing is attached herewith as an XML file named G11718-00471-SSS_Seqlisting.xml, created on November 14, 2023, and approximately 100 kilobytes in size. The contents of the above-mentioned file are hereby incorporated by reference in their entirety. Technical Field

[0005] The present invention relates generally to the field of oncology and, more particularly, to the treatment of cancer. Background Art

[0006] The incidence of cancer in humans and animals, and its impact on mortality, means there is a continuing need for new, effective anti-tumor drugs. Eliminating a tumor, reducing its size, or decreasing the number of cancer cells circulating in the blood or lymphatic system could have multiple benefits: reducing pain or discomfort, preventing metastasis, facilitating surgical intervention, and, importantly, prolonging life.

[0007] Various attempts have been made to help the immune system fight tumors. In the late 19th century, one early approach involved global stimulation of the immune system, for example, by administering bacteria (live or killed) to elicit a global immune response that would also be directed against the tumor.

[0008] Recent approaches aim to help the immune system specifically recognize tumor-specific antigens (TSAs) (or tumor-associated antigens, TAAs), which involve administering tumor-specific antigens to a subject, typically in combination with an adjuvant. However, a lack of a robust immune response to TAAs is often observed in cancer. One of the factors that contributes to a weak response to TAAs is the induction of inhibitory pathways / signals (commonly referred to as "immune checkpoints") that suppress the immune response. While such inhibitory signals are important for maintaining self-tolerance and for protecting tissues from damage when the immune system responds to pathogen infection, the inhibitory signals may also reduce the beneficial responses that the body might otherwise make to tumorigenesis.

[0009] A new era of treatment has emerged with the use of immune checkpoint inhibitors or blockers (ICBs) targeting inhibitory T-cell receptors such as CTLA-4, PD-L1, and PD-1 (Marabelle, OncoImmunology, 2016). This emerging field even garnered the 2018 Nobel Prize in Medicine. These immunotherapies have demonstrated promising clinical responses in several advanced cancers, including lung cancer (Reck, NEJM, 2016), melanoma (Robert, NEJM, 2011), genitourinary cancers (Motzer, NEJM, 2018), and head and neck cancers (Ferris, NEJM, 2016). However, primary resistance rates in patients with non-small cell lung cancer (NSCLC) range from 35% to 44%, while secondary resistance rates approach 100% (Reck, NEJM, 2016).

[0010] Several tumors are considered immune “cold” tumors, i.e., tumors that are unlikely to elicit a strong immune response and generally respond poorly to cancer immunotherapies such as ICB therapy (see, e.g., Bonaventura et al., “Cold Tumors: A Therapeutic Challenge for Immunotherapy.” Frontiers in Immunology, Vol. 10, 168 (2019). A notable feature of these tumors is the absence of tumor infiltration by tumor antigen-specific immune cells, such as tumor infiltrating lymphocytes (TILs).

[0011] Therefore, there is a need to develop new approaches to induce or stimulate immune responses against tumors, particularly immune-cold tumors and / or tumors that are resistant to immunotherapy.

[0012] This specification refers to various documents, the contents of which are incorporated herein by reference in their entireties. Summary of the Invention

[0013] In various aspects and embodiments, the present disclosure provides the following items 1 to 78:

[0014] 1. A method for (i) enhancing an anti-tumor immune response in a subject suffering from a cancer expressing Sortilin and / or (ii) treating a subject suffering from a cancer expressing Sortilin that is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound is of formula A-(B) n,in

[0015] A is a peptide compound of 30 residues or less comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13:

[0016] X1X2X3X4X5GVX6AKAGVX7NX8FKSESY (SEQ ID NO:1)

[0017] (X9) n GVX 10 AKAGVX 11 NX 12 FKSESY (SEQ ID NO: 2)

[0018] YKX 13 LRRX 14 APRWX 15 PLRDPALRX 16 X 17 L (SEQ ID NO: 3)

[0019] YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 L (SEQ ID NO:4)

[0020] IKLSGGVQAKAGVINMDKSESM (SEQ ID NO:5)

[0021] IKLSGGVQAKAGVINMFKSESY (SEQ ID NO:6)

[0022] IKLSGGVQAKAGVINMFKSESYK (SEQ ID NO:7)

[0023] GVQAKAGVINMFKSESY (SEQ ID NO:8)

[0024] GVRAKAGVRNMFKSESY (SEQ ID NO:9)

[0025] GVRAKAGVRN(Nle)FKSESY (SEQ ID NO:10)

[0026] YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO:11)

[0027] YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO:12)

[0028] YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO:13)

[0029] in

[0030] 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 independently selected from any amino acid;

[0031] X 16 、X 17 、X 20 and X 21 independently selected from Q, P, Y, I and L;

[0032] n is 0, 1, 2, 3, 4, or 5;

[0033] When X9 is present more than once, each of said X9 is independently selected from any amino acid;

[0034] When X 19 When present more than once, each of said X9 is independently selected from any amino acid,

[0035] Optionally, the peptide compound is cyclic,

[0036] B is at least one therapeutic agent, wherein B is linked to A directly or via a linker.

[0037] 2. The method according to item 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: 1-13.

[0038] 3. The method according to item 1 or 2, wherein the peptide compound comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-13, and further comprises 1 to 3 additional amino acids at its amino and / or carboxyl terminus.

[0039] 4. The method according to item 3, wherein the peptide compound comprises a cysteine ​​residue at its amino terminus and / or carboxyl terminus.

[0040] 5. The method according to item 4, wherein the peptide compound comprises or consists of one of the following amino acid sequences:

[0041] Z1X1X2X3X4X5GVX6AKAGVX7NX8FKSESYZ2 (SEQ ID NO:34)

[0042] Z1(X9) n GVX 10 AKAGVX 11 NX 12 FKSESYZ2 (SEQ ID NO:35)

[0043] ZlUT 13 LRRX 14 APRWX 15 PLRDPALRX 16 X 17 LZ2 (SEQ ID NO:36)

[0044] Z1YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 LZ2 (SEQ ID NO:37)

[0045] Z1IKLSGGVQAKAGVINMDKSESMZ2 (SEQ ID NO:38)

[0046] Z1IKLSGGVQAKAGVINMFKSESYZ2 (SEQ ID NO:39)

[0047] Z1IKLSGGVQAKAGVINMFKSESYKZ2 (SEQ ID NO:40)

[0048] Z1GVQAKAGVINMFKSESYZ2 (SEQ ID NO:41)

[0049] Z1GVRAKAGVRNMFKSESYZ2 (SEQ ID NO:42)

[0050] Z1GVRAKAGVRN(Nle)FKSESYZ2 (SEQ ID NO:43)

[0051] Z1YKSLRRKAPRWDAPLRDPALRQLLZ2 (SEQ ID NO:44)

[0052] Z1YKSLRRKAPRWDAYLRDPALRQLLZ2 (SEQ ID NO:45)

[0053] Z1YKSLRRKAPRWDAYLRDPALRPLL Z2 (SEQ ID NO:46),

[0054] Where X1-X 21 As defined in item 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.

[0055] 6. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 2.

[0056] 7. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 4.

[0057] 8. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0058] 9. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0059] 10. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0060] 11. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0061] 12. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0062] 13. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0063] 14. The method according to item 13, wherein the peptide compound comprises or consists of the amino acid sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 47).

[0064] 15. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0065] 16. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0066] 17. The method according to item 1 or 5, wherein the peptide compound comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0067] 18. The method according to any one of items 1 to 17, wherein the peptide compound comprises at least one modification group at its amino terminus and / or carboxyl terminus.

[0068] 19. The method according to item 17, wherein the at least one modifying group is acetyl or succinyl.

[0069] 20. The method according to item 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:

[0070] Acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14)

[0071] Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO:15)

[0072] Acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16)

[0073] Acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17)

[0074] Acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18)

[0075] Acetyl-GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO:48).

[0076] 21. The method according to any one of items 1 to 20, wherein B is linked to A at a free amine of the peptide compound, at the N-terminal position of the peptide compound, at a free -SH group of the peptide compound and / or at a free carboxyl group of the peptide compound.

[0077] 22. The method according to any one of items 1 to 21, wherein B is linked to A via a linker.

[0078] 23. The method according to any one of items 1 to 22, wherein the conjugate is represented by formula (LIII) or (LIV):

[0079] GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(LIII) (SEQ ID NO:22);

[0080] Acetyl-GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(LIV) (SEQ ID NO: 23);

[0081] Among them J 1 and J 2 Each is independently a therapeutic agent linked to a lysine (K) residue.

[0082] 24. The method according to any one of items 1 to 23, wherein the therapeutic agent is an anti-tumor agent, such as a radionuclide or a chemotherapeutic agent.

[0083] 25. The method according to item 24, wherein the chemotherapeutic agent is a taxane.

[0084] 26. The method according to item 25, wherein the chemotherapeutic agent is docetaxel.

[0085] 27. The method according to any one of items 1 to 26, further comprising treating the subject with immunotherapy.

[0086] 28. The method according to any one of items 1 to 27, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0087] 29. The method according to item 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.

[0088] 30. The method according to item 28 or 29, wherein the ICI is a blocking antibody.

[0089] 31. The method according to item 28 or 29, wherein the ICI is a PD-L1 inhibitor.

[0090] 32. The method according to any one of items 1 to 31, wherein the cancer is an immunologically cold cancer.

[0091] 33. The method according to any one of items 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.

[0092] 34. Use of a conjugate compound as defined in any one of items 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.

[0093] 35. Use of a conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for (i) enhancing the 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.

[0094] 36. The use according to item 34 or 35, wherein the conjugate compound, a pharmaceutically acceptable salt thereof or a drug is used in combination with immunotherapy.

[0095] 37. Use according to any one of items 34 to 36, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0096] 38. The use according to item 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.

[0097] 39. Use according to item 37 or 38, wherein the ICI is a blocking antibody.

[0098] 40. The use according to item 38 or 39, wherein the ICI is a PD-L1 inhibitor.

[0099] 41. Use according to any one of items 34 to 40, wherein the cancer is an immunologically cold cancer.

[0100] 42. Use according to any one of items 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.

[0101] 43. A conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, for use in (i) enhancing the 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.

[0102] 44. The conjugate compound for use according to item 43, or a pharmaceutically acceptable salt thereof, wherein the peptide compound is used in combination with immunotherapy.

[0103] 45. The conjugate compound for use according to item 43 or 44, or a pharmaceutically acceptable salt thereof, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0104] 46. ​​The conjugate compound for use according to item 45, or a pharmaceutically acceptable salt thereof, 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.

[0105] 47. The conjugate compound for use according to item 45 or 46, or a pharmaceutically acceptable salt thereof, wherein the ICI is a blocking antibody.

[0106] 48. The conjugate compound for use according to item 46 or 47, or a pharmaceutically acceptable salt thereof, wherein the ICI is a PD-L1 inhibitor.

[0107] 49. The conjugate compound for use according to any one of items 43 to 48, or a pharmaceutically acceptable salt thereof, wherein the cancer is an immunologically cold cancer.

[0108] 50. The conjugate compound for use according to any one of items 43 to 49, or a pharmaceutically acceptable salt thereof, wherein the cancer is brain cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, thyroid cancer, lung cancer, prostate cancer or melanoma.

[0109] 51. A method for treating a Sortilin-expressing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, in combination with immunotherapy.

[0110] 52. The method according to item 51, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0111] 53. The method according to item 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.

[0112] 54. The method according to item 52 or 53, wherein the ICI is a blocking antibody.

[0113] 55. The method according to item 53 or 54, wherein the ICI is a PD-L1 inhibitor.

[0114] 56. The method according to any one of items 51 to 55, wherein the cancer is an immunologically cold cancer.

[0115] 57. The method according to any one of items 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.

[0116] 58. The method according to any one of items 51 to 57, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in different compositions.

[0117] 59. The method according to any one of items 51 to 57, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in the same composition.

[0118] 60. Use of a conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, in combination with immunotherapy for the treatment of a cancer expressing Sortilin.

[0119] 61. Use of a conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, in combination with immunotherapy for the preparation of a medicament for the treatment of a cancer expressing Sortilin.

[0120] 62. The use according to item 60 or 61, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0121] 63. The use according to item 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.

[0122] 64. Use according to item 62 or 63, wherein the ICI is a blocking antibody.

[0123] 65. The use according to item 63 or 64, wherein the ICI is a PD-L1 inhibitor.

[0124] 66. Use according to any one of items 60 to 65, wherein the cancer is an immunologically cold cancer.

[0125] 67. Use according to any one of items 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.

[0126] 68. The use according to any one of items 61 to 67, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in different compositions.

[0127] 69. The use according to any one of items 61 to 67, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in the same composition.

[0128] 70. A combination therapy comprising a conjugate compound as defined in any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, and an immunotherapy, for use in treating a cancer expressing Sortilin.

[0129] 71. The combination therapy for use according to item 70, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

[0130] 72. The combination therapy for use according to item 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.

[0131] 73. The combination therapy for use according to item 71 or 72, wherein the ICI is a blocking antibody.

[0132] 74. The combination therapy for use according to item 72 or 73, wherein the ICI is a PD-L1 inhibitor.

[0133] 75. The combination therapy for use according to any one of items 71 to 74, wherein the cancer is an immunologically cold cancer.

[0134] 76. The combination therapy for use according to any one of items 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.

[0135] 77. The combination therapy for use according to any one of items 70 to 76, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in different compositions.

[0136] 78. The combination therapy for use according to any one of items 70 to 76, wherein the conjugate compound or a pharmaceutically acceptable salt thereof and the immunotherapy are in the same composition.

[0137] Other objects, advantages and features of the present invention will become more apparent from a reading of the following non-restrictive description of specific embodiments of the invention, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] In the attached figure:

[0139] Figure 1A -I shows the anti-tumor activity of TH1902 in immunosuppressive MDA-MB-231TNBC derived xenograft model that lasts and is prolonged.As previously described, generate in vivo MDA-MB-231TNBC xenograft model in immunocompromised nude mice [5].With MTD be 15mg / kg / wk of docetaxel or with 35mg / kg / wk of TH1902 intravenous injection and carry out mouse treatment, and (i) after three cycles or for TH1902 (ii) six cycles after four days stop.Other group is processed (iii) six cycles with TH1902, then stops three cycles. Figure 1A Tumor growth was monitored on the indicated days as described in the Examples. Data are presented as mean ± SEM (three mice / group). Representative sections of the tumors were stained with H&E ( Figure 1B ), or for SORT1, Ki67, STING, and CD45 ( Figure 1C ) and stained as described in Example 1. Figure 1D :By monitoring CD31 - / PAS + Angiogenesis mimics were assessed by staining, while normal vessels were assessed by CD31 + / PAS + Dyeing to illustrate. Figure 1E : The body weight of mice was monitored as described in Example 1. The body weight of mice is expressed as a percentage of the initial body weight at the start of treatment (day 0). Data are expressed as mean ± SEM (three mice / group). Figure 1FG: The different effects of TH1902 and docetaxel on the expression of p21 and p53 in MDA-MB-231 cells. MDA-MB-231 cells were treated with vehicle (DMSO) or 50nM docetaxel or TH1902 for 5 minutes and then incubated in fresh complete medium for 96 hours. Cell lysates were harvested as described in Example 1 and representative immunoblotting was performed with anti-p21, anti-p53 and anti-GAPDH antibodies ( Figure 1F ), and protein expression was quantified using densitometry ( Figure 1F ). Figure 1H and I: PD-L1 quantification in MDA-MB-231 / Luc tumors in vivo using QuPath software. Figure 1H :% of PD-L1 positive area; Figure 1I : % of PD-L1 positive cells.

[0140] Figure 2A -C shows SORT1 expression in melanoma tissue and cell line models. Figure 2A : SORT1 expression was assessed by immunohistochemistry in healthy tissue and tissue microarrays of clinically annotated stage II-IV melanomas. Figure 2B : 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 : SORT1 expression levels were assessed by Western blot analysis in cell lysates (20 μg) from different cancer cell lines. B16-F10, murine B16-F10 melanoma cells; SKMEL, human SK-MEL-28 melanoma cells; A375, human A375 melanoma cells; MDA, human TNBC-derived MDA-MB-231 cells.

[0141] Figure 3A -G shows that TH1902 exhibits anti-proliferative and apoptotic activities in vitro and induces senescence of SORT1-positive melanoma cells. Figure 3A ) and B16-F10( Figure 3B ) melanoma cells to assess cell proliferation in response to docetaxel or TH1902, as described in Example 1, and extract the IC of each test article. 50 value( Figure 3C ). Figure 3D : Apoptosis was assessed in B16-F10 melanoma cells after treatment with the indicated concentrations of docetaxel (black bars) and TH1902 (grey bars). Figure 3E: Fluorescence microscopy was used to assess cellular senescence in B16-F10 melanoma cells after treatment with docetaxel, TH1902, or etoposide (as a positive control for senescence induction), and representative images are shown. Figure 3F : Senescence-associated β-galactosidase activity was quantified as described in Example 1 (n=3). Figure 3G : Changes in cell morphology were assessed using crystal violet staining of cells after treatment with vehicle (DMSO), 100 nM docetaxel, or 50 nM TH1902 (equivalent docetaxel content).

[0142] Figure 4A -E shows the infiltration of leukocytes within B16-F10 tumors treated with vehicle, docetaxel, or TH1902. Figure 4A : Tumor growth in syngeneic mice treated with vehicle, 15 mg / kg / wk docetaxel (MTD), or 35 mg / kg / wk TH1902 (equivalent docetaxel content). Data are presented as mean ± SEM (9 mice / group for vehicle and docetaxel, 10 mice / group for TH1902). Figure 4B : B16-F10 melanoma was excised and photographed. Figure 4C : The excised tumors were fixed with formalin and processed for immunohistochemical analysis. The top layer of the image was stained with hematoxylin and eosin; the bottom layer was immunohistochemically evaluated using a monoclonal antibody against CD45 (pan-immunocytes). Representative images of the whole and magnified tumors of both stainings are shown (black scale bar = 2 mm, white scale bar = 100 μm). Figure 4D The amount of CD45-stained area was compared between the three groups of mice by one-way ANOVA followed by Tukey's multiple comparisons test. The positive staining area of ​​leukocytes in animals treated with TH1902 was significantly greater than that in animals treated with vehicle or docetaxel. Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, n = 4 tumors analyzed per group). Figure 4E Figure 2: Mouse weights in syngeneic mice treated with vehicle, 15 mg / kg / wk docetaxel (MTD), or 35 mg / kg / wk TH1902 (equivalent to docetaxel). Mouse weights are expressed as a percentage of initial body weight at the start of treatment (day 0). Data are presented as mean ± SEM (9 mice / group for vehicle and docetaxel, 10 mice / group for TH1902).

[0143] Figure 5A-D shows the effects of docetaxel and TH1902 on the levels of tumor-infiltrating lymphocytes and macrophages within syngeneic tumors. Figure 5A : Each row of micrographs depicts cells from B16-F10 tumors in animals treated with vehicle, docetaxel, or TH1902 ( Figure 4A -E same samples). Each column includes 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 from primary tumors (white scale bar = 100 μm). Figure 5B :From Figure 5A Quantification of IHC staining of . Expression was compared using one-way ANOVA with Tukey's multiple comparison test. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, n = 4 tumors analyzed per group). Figure 5C -D is a graph showing the quantification of immune cell infiltration in B16-F10 xenograft tumors treated with docetaxel and TH1902. Figure 5C :Natural killer (NK) cells (CD161c + )and Figure 5D :Tumor-associated macrophages (TAM), including M1 (CD68 + ) and M2(CD206 + ) macrophages.

[0144] Figure 6A -B shows the effects of docetaxel and TH1902 on the expression of tumor markers for immune-stimulated apoptosis. Figure 6A : Each row of micrographs depicts cells from B16-F10 tumors in animals treated with vehicle, docetaxel, or TH1902 (same samples as in FIG4 ). Each column includes representative IHC images of cleaved caspase-3, perforin, and granzyme B, markers involved in immune-stimulated apoptosis (white scale bar = 100 μm). Figure 6B :From Figure 6A Quantification of IHC staining of . Expression was compared using one-way ANOVA with Tukey's multiple comparison test. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, n = 4 tumors analyzed per group).

[0145] Figure 7A -D shows the effect of TH1902 combined with checkpoint inhibitors on tumor growth and mouse survival. B16-F10 cells were subcutaneously implanted into immunocompetent C57BL / 6 mice. Figure 7AEffect of docetaxel, TH1902, and anti-PD-L1, alone or in combination, on B16-F10 tumor growth. Mice were treated weekly with intravenous administration of vehicle, docetaxel (7.5 mg / kg), or TH1902 (17.5 mg / kg; equivalent dose of docetaxel) or biweekly with intraperitoneal administration of 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 of treatment. Data are presented as mean ± SEM (n = 8 mice / group). Figure 7B and C: Effects of increasing doses of TH1902 and anti-PD-L1 alone or in combination on survival in B16-F10 tumor-bearing mice ( Figure 7B ) and tumor growth ( Figure 7C ). Mice were treated weekly with intravenous administration of vehicle and TH1902 (4.37, 8.75, 17.5 mg / kg) or biweekly with intraperitoneal administration of anti-PD-L1 (9 mg / kg) and control isotype (9 mg / kg), alone or in combination, for continuous cycles of treatment until one of the defined endpoints as described in Example 1 was reached. Kaplan-Meier curves were plotted to estimate mouse survival and expressed as the probability of survival in percentage (top graph), while tumor growth curves were plotted until one individual in a given group reached the tumor size endpoint (tumor > 2,000 mm 3 ; bottom panel). Data are presented as mean ± SEM (n = 6 mice / group). Figure 7D : Mouse body weight is expressed as a percentage of the initial body weight at the start of treatment (day 0). Data are expressed as mean ± SEM (n = 8 mice / group).

[0146] Figure 8 Described are the effects of various checkpoint inhibitors on B16-F10 melanoma tumor growth (corresponding to Figure 1A , from Ueha et al., Cancer Immunol Res (2015) 3(6):631–640). Mice bearing B16-F10 melanoma tumors were injected intraperitoneally (ip) with anti-CD4 mAb (200 mg / mouse) on days 5 and 9 after tumor inoculation and with anti-immune checkpoint mAb (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.

[0147] Figure 9A-F shows that TH1902 induces the downstream effectors of the STING pathway and the 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 and then incubated in fresh complete medium for 96 hours. Cell lysates were harvested as described in Example 1 and immunoblotted with anti-STING and anti-GAPDH antibodies ( Figure 9A ), and protein expression was quantified using densitometry ( Figure 9B ). Figure 9C : Lysates were further processed for expression of STING downstream effectors in the presence of 100 nM docetaxel or TH1902 and subjected to density quantification ( Figure 9D ). Figure 9E : Total RNA was extracted from cells treated with 100 nM docetaxel or TH1902 or left untreated for 5 minutes and then incubated in fresh complete medium for 24 hours, and gene expression of IL-6 and TNFα was assessed using RT-qPCR. Figure 9F : Immunophenotyping for MHC-I and PD-L1 cell surface expression was performed by flow cytometry as described in Example 1.

[0148] Figure 10A -B shows that TNFα, but not IL-6, triggers 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. Figure 10A ) or IL-6 treatment ( Figure 10B ) when PD-L1 and MHC-I cell surface expression.

[0149] Figure 11A -B depicts a demonstration of the efficacy of the checkpoint inhibitor anti-PD1 ( Figure 11A ) and anti-PD-L1( Figure 11B ) treatment on the LL / 2 (Lewis Lung) lung cancer model (from https: / / drugdevelopment.labcorp.com / industry-solutions / oncology / preclinical / tumor-spotlights / ll-2-an-immunosuppressive-murine-tumor-model.html, Figure 5).

[0150] Figure 11CThe effects of TH1902 and docetaxel on LL / 2 lung cancer xenografts are shown. LL / 2 cells were subcutaneously implanted into the back region of C57BL / 6 mice and treated after 3 days. Tumor volume was measured after weekly administration of vehicle, docetaxel (15 mg / kg) or TH1902 (35 mg / kg). Data are expressed as mean ± SEM (n=7 mice / group. Exponential curve analysis (additional sum of squares F test) shows that there are significant differences between all curves.

[0151] Figure 12 The amino acid sequence of human Sortilin-1 (SORT1, UniProtKB Accession No. Q99523) is depicted. DETAILED DESCRIPTION

[0152] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0153] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise noted.

[0154] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values ​​within the range are also incorporated into the specification as if they were individually recited herein.

[0155] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0156] The use of any and all examples, or exemplary language ("such as," "eg," etc.) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0157] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0158] As used herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value, or encompasses values ​​close to the stated value, for example, within 10% of the stated value (or range of values).

[0159] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0160] The scope of the claims should not be limited by the preferred embodiments listed in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[0161] The present invention encompasses any and all combinations and subcombinations of the embodiments and features disclosed herein.

[0162] In the studies described herein, the inventors have demonstrated in a murine model of immune-cold tumors that a peptide targeting Sortilin conjugated to a chemotherapeutic agent (docetaxel) can induce anti-tumor immune cell infiltration in tumors and inhibit tumor growth, and enhance the anti-tumor response of immune checkpoint inhibitors.

[0163] Therefore, in the first aspect, the present disclosure provides a method for (i) enhancing the anti-tumor immune response of a subject with cancer and / or (ii) treating a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy), the method comprising administering an effective amount of a conjugate compound as defined herein or a pharmaceutically acceptable salt thereof to the subject. The present disclosure also provides the use of a conjugate compound as defined herein or a pharmaceutically acceptable salt thereof, which is used for (i) enhancing the anti-tumor immune response of a subject with cancer and / or (ii) treating a subject with a cancer 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, which is used to prepare a medicament for (i) enhancing the anti-tumor immune response of a subject with cancer and / or (ii) treating a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). The present disclosure also provides a conjugate compound as defined herein, or a pharmaceutically acceptable salt thereof, for use in (i) enhancing an anti-tumor immune response in a subject having cancer and / or (ii) treating a subject having cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0164] In one embodiment, the method or use is used to enhance the anti-tumor immune response of a subject having cancer. In another embodiment, the method or use is used to treat a subject having a cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). In another embodiment, the method or use is used to enhance the anti-tumor immune response of a subject having a cancer that is resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0165] Conjugates suitable for use in the methods and uses disclosed herein are conjugates comprising a therapeutic agent, such as an anti-tumor agent (e.g., a chemotherapeutic agent), conjugated to an agent capable of binding to sortilin and being internalized by tumor cells, such as a peptide, so as to deliver the anti-tumor agent to the tumor cells. In one embodiment, the conjugate (or conjugate compound) comprises 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 or antigen-binding fragments thereof include anti-sortilin monoclonal antibody clone 2D8-E3 (Ghaemimanesh F. et al., Monoclonal Antibodies in Immunodiagn Immunother 2015;34(6):390-5), antibodies and antigen-binding fragments thereof are disclosed in PCT Publication Nos. WO 2016 / 164637, WO 2017 / 009327, WO 2019 / 016247, WO 2020 / 014617, WO 2020 / 252066, WO 2021 / 116290, WO 2021 / 263279, WO 2022 / 261648, and WO 2023 / 122766, the contents of which are incorporated herein by reference.

[0166] Peptide compounds that bind to sortilin are disclosed in PCT Publication Nos. WO 2017 / 088058, WO 2018 / 213928, WO 2020 / 037434, WO 2021 / 078833, WO 2021 / 028404, and WO 2023 / 165476, the contents of which are incorporated herein by reference.

[0167] In one embodiment, the conjugate (or conjugate compound) is an anti-tumor agent-peptide compound conjugate, as described in PCT Publication Nos. WO 2017 / 088058, WO 2018 / 213928, and WO 2020 / 037434.

[0168] As used herein, the term "sortilin" or "sortilin receptor" refers to a neuronal type 1 membrane glycoprotein encoded by the SORT1 gene, which belongs to the vacuolar protein sorting 10 protein (Vps10) family of receptors. Sortilin (also known as neurotensin receptor 3; UniProtKB accession number Q99523) is expressed or overexpressed in a variety of cancers including, for example, ovarian cancer, breast cancer, colon cancer, and prostate cancer. The encoded preproprotein (residues 34-831, residues 1-33 correspond to the signal peptide) is proteolytically processed by furin (or other homologous proteases) after amino acid 77 to generate a mature receptor (residues 78-831) with a molecular weight of approximately 100-110 kDa. The amino acid residues of sortilin referred to herein correspond to the positions in the full-length form (i.e., UniProtKB accession number Q99523, Figure 12 ).

[0169] In one embodiment, the conjugate compound has the formula A-(B) n ,in

[0170] A is a peptide compound comprising an amino acid sequence having at least 60% sequence identity to one of the sequences of Formulae (I)-(XIII):

[0171] X1X2X3X4X5GVX6AKAGVX7NX8FKSESY (I) (SEQ ID NO:1)

[0172] (X9) n GVX 10 AKAGVX 11 NX 12 FKSESY (II) (SEQ ID NO: 2)

[0173] YKX 13 LRRX 14 APRWX 15 PLRDPALRX 16 X 17 L(III) (SEQ ID NO:3)

[0174] YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 L (IV) (SEQ ID NO:4)

[0175] IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO:5)

[0176] IKLSGGVQAKAGVINMFKSESY (VI) (SEQ ID NO:6)

[0177] IKLSGGVQAKAGVINMFKSESYK (VII)(SEQ ID NO:7)

[0178] GVQAKAGVINMFKSESY (VIII)(SEQ ID NO:8)

[0179] GVRAKAGVRNMFKSESY (IX) (SEQ ID NO:9)

[0180] GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO:10)

[0181] YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO:11)

[0182] YKSLRRKAPRWDAYLRDPALRQLL (XII)(SEQ ID NO:12)

[0183] YKSLRRKAPRWDAYLRDPALRPLL (XIII)(SEQ ID NO:13)

[0184] in

[0185] 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 Independently selected from any amino acid; X 16 、X 17 、X 20 and X 21 are independently selected from Q, P, Y, I and L; n is an integer from 1 to 10, such as 1, 2, 3, 4 or 5; when X9 is present more than once, each of said X9 is independently selected from any amino acid; when X 19 When present more than once, each of said X9 is independently selected from any amino acid, optionally said peptide compound is cyclic, and wherein said peptide compound binds to Sortilin.

[0186] B is at least one therapeutic agent, such as an anti-tumor agent, wherein B is optionally linked to A directly or via a linker at a free amine of the peptide compound, at the N-terminal position of the peptide compound, at a free -SH group of the peptide compound, or at a free carboxyl group of the peptide compound,

[0187] or a pharmaceutically acceptable salt thereof.

[0188] The term "amino acid" refers to common natural (genetically encoded) or synthetic amino acids and their common derivatives known to those skilled 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, "non-standard," "non-natural," or "unusual" refers to a wide selection of non-natural, rare, or synthetic amino acids, such as those 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-α-amino acids and D-amino acids. In one embodiment, the peptide compound comprises only natural amino acids. In another embodiment, the peptide compound comprises one or more non-natural or synthetic amino acids, such as D-amino acids.

[0189] As used herein, the expression "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. In order to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence to optimally align with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at said position. The percentage of identity between the 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 x 100%). In one embodiment, the two sequences are of the same length. The determination of the percentage of identity between the two sequences can also be accomplished using a mathematical algorithm. A BLAST protein search can be performed using the XBLAST program parameter set (e.g., score -50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be utilized. Alternatively, PSI-BLAST can be used for the iterative search (the same) that performs the distance relationship between the detection molecules. When utilizing BLAST, there is BLAST and PSI-Blast program in the gap, the default parameters of the corresponding program (e.g., XBLAST and NBLAST) can be used (see, e.g., NCBI website). Another preferred non-limiting example of the mathematical algorithm for sequence comparison is Myers and Miller, 1988, " Computer Applications in Biological Sciences (CABIOS) " 4:11-17 algorithm. This algorithm is incorporated into the ALIGN program (version 2.0), which is a part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, PAM120 weight residue table, gap length penalty 12 and gap penalty 4 can be used. When allowing or not allowing gaps, the technology similar to that described above can be used to determine the identity percentage between the two sequences. When calculating the identity percentage, usually only exact matching is counted.

[0190] The expression "pharmaceutically acceptable" means compatible with treatment of a subject, such as an animal or a human. Also provided herein are pharmaceutically acceptable salts of the conjugate compounds described herein. The expression "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt that is suitable for or compatible with treatment of a subject, such as an animal or a human. As used herein, the expression "pharmaceutically acceptable acid addition salt" means any non-toxic organic or inorganic salt of any compound of the present disclosure or any intermediate thereof. Illustrative inorganic acids that form suitable salts include hydrochlorides, hydrobromides, sulfates, and phosphates, as well as metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, 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-toluenesulfonate and methanesulfonic acid. Monoacid salts or diacid salts can be formed, and such salts can exist in hydrated, solvated or substantially anhydrous forms. Generally, acid addition salts of the compounds of the present disclosure are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than their free base forms. Those skilled in the art will appreciate the selection of appropriate salts. Other non-pharmaceutically acceptable salts, such as oxalates, can be used, for example, to isolate the compounds of the present disclosure for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts. As used herein, the expression "pharmaceutically acceptable base addition salt" means any non-toxic organic or inorganic base addition salt of any acid compound of the present disclosure or any intermediate thereof. Acidic compounds of the present disclosure that can form base addition salts include, for example, wherein CO2H is a functional group. Illustrative inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline or ammonia. Those skilled in the art will appreciate the selection of appropriate salts.Other non-pharmaceutically acceptable base addition salts may be useful, for example, in isolating a compound or conjugate compound of the disclosure for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0191] In one embodiment, the peptide compound comprises or consists of a sequence of any one of Formulas (I)-(XIII). In one embodiment, the peptide compound comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1-13. In embodiments, the peptide compound comprises 50, 45, 40, 35, 30, 25, or 20 amino acids or less.

[0192] In embodiments, the peptide 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 peptide compound represented by Formula (I) or SEQ ID NO: 1, wherein the peptide compound binds to Sortilin.

[0193] In embodiments, the peptide 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 peptide compound represented by Formula (II) or SEQ ID NO: 2, wherein the peptide compound binds to Sortilin.

[0194] In embodiments, the peptide 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 peptide compound represented by Formula (III) or SEQ ID NO: 3, wherein the peptide compound binds to Sortilin.

[0195] In embodiments, the peptide 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 peptide compound represented by Formula (IV) or SEQ ID NO: 4, wherein the peptide compound binds to Sortilin.

[0196] In embodiments, the peptide 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 peptide compound represented by Formula (V) or SEQ ID NO: 5, wherein the peptide compound binds to Sortilin.

[0197] In embodiments, the peptide 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 peptide compound represented by Formula (VI) or SEQ ID NO: 6, wherein the peptide compound binds to Sortilin.

[0198] In embodiments, the peptide 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 peptide compound represented by Formula (VII) or SEQ ID NO: 7, wherein the peptide compound binds to Sortilin.

[0199] In embodiments, the peptide 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 peptide compound represented by Formula (VIII) or SEQ ID NO: 8, wherein the peptide compound binds to Sortilin.

[0200] In embodiments, the peptide 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 peptide compound represented by Formula (IX) or SEQ ID NO: 9, wherein the peptide compound binds to Sortilin.

[0201] In embodiments, the peptide 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 the peptide compound represented by Formula (X) or SEQ ID NO: 10, wherein the peptide compound binds to Sortilin.

[0202] In embodiments, the peptide 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 peptide compound represented by Formula (XI) or SEQ ID NO: 11, wherein the peptide compound binds to Sortilin.

[0203] In embodiments, the peptide 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 the peptide compound represented by Formula (XII) or SEQ ID NO: 12, wherein the peptide compound binds to Sortilin.

[0204] In embodiments, the peptide 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 peptide compound represented by Formula (XIII) or SEQ ID NO: 13, wherein the peptide compound binds to Sortilin.

[0205] In one embodiment, the peptide compound comprises 30, 25 or 20 residues or less and comprises the sequence GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 10). In another embodiment, the peptide compound comprises 30, 25 or 20 residues or less and comprises the sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO: 31).

[0206] In one embodiment, at least one modifying group is attached to the peptide compound at the N-terminus and / or the C-terminus. In one embodiment, the peptide compound comprises a modifying group at the N-terminus. In one embodiment, the peptide compound comprises a modifying group at the C-terminus. Such modifying groups can be used to protect the peptide compound from modification or degradation (e.g., protease degradation). In one embodiment, the amino terminal modifying group is C1-C 16 or C3-C 16 Acyl (straight or branched, saturated or unsaturated), in another embodiment, is a saturated C1-C6 acyl (straight or branched) or an unsaturated C3-C6 acyl (straight or branched). In another embodiment, the amino terminal modification group is an acetyl (CH3-CO-, Ac) or a succinyl (CO-CH2-CH2-CO-). The carboxyl terminal modification group can be, for example, a hydroxylamine (NHOH) (-C(=O)-NHOH) connected to a carboxyl group, or an amine (-C(=O)-NRR') connected to a carboxyl group, the amine being a primary amine, a secondary amine or a tertiary amine, and preferably the amine is an aliphatic amine preferably having one to ten carbon atoms, such as methylamine, isobutylamine, isovaleramide or cyclohexylamine, an aromatic amine or an arylalkylamine, such as aniline, naphthylamine, benzylamine, cinnamylamine or phenylethylamine, preferably the amine is -NH2.

[0207] In one embodiment, a succinyl group is attached to the peptide compound. For example, the peptide compound has a sequence of succinyl-IKLSGGVQAKAGVINMFKSESY, corresponding to SEQ ID NO: 6, and has a succinyl group attached to it at the N-terminus.

[0208] In one embodiment, an acetyl group is attached to the peptide compound. For example, the peptide compound has the sequence of acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14). For example, the peptide compound has the sequence of acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15). For example, the peptide compound has the sequence of acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16). For example, the peptide compound has the sequence of acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17). For example, the peptide compound has the sequence of acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18).

[0209] In one embodiment, the peptide compound can be modified at the C-terminus and / or N-terminus by adding one or more (e.g., 1 to 5 or 1 to 3) amino acid residues to obtain or increase a preferential binding site at the peptide end. 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 at the N-terminus and / or C-terminus of the peptide. In one embodiment, the peptide compound is modified by adding cysteine ​​at the C-terminus.

[0210] Thus, in an embodiment, the peptide compound comprises or consists of one of the following amino acid sequences:

[0211] Z1X1X2X3X4X5GVX6AKAGVX7NX8FKSESYZ2 (SEQ ID NO:34)

[0212] Z1(X9) n GVX 10 AKAGVX 11 NX 12 FKSESYZ2 (SEQ ID NO:35)

[0213] ZlUT 13 LRRX 14 APRWX 15 PLRDPALRX 16 X 17 LZ2 (SEQ ID NO: 36)

[0214] Z1YKX 18 LRR(X 19 ) N PLRDPALRX 20 X 21 LZ2 (SEQ ID NO:37)

[0215] Z1IKLSGGVQAKAGVINMDKSESMZ2 (SEQ ID NO:38)

[0216] Z1IKLSGGVQAKAGVINMFKSESYZ2 (SEQ ID NO:39)

[0217] Z1IKLSGGVQAKAGVINMFKSESYKZ2 (SEQ ID NO:40)

[0218] Z1GVQAKAGVINMFKSESYZ2 (SEQ ID NO:41)

[0219] Z1GVRAKAGVRNMFKSESYZ2 (SEQ ID NO:42)

[0220] Z1GVRAKAGVRN(Nle)FKSESYZ2 (SEQ ID NO:43)

[0221] Z1YKSLRRKAPRWDAPLRDPALRQLLZ2 (SEQ ID NO:44)

[0222] Z1YKSLRRKAPRWDAYLRDPALRQLLZ2 (SEQ ID NO:45)

[0223] Z1YKSLRRKAPRWDAYLRDPALRPLLZ2 (SEQ ID NO:46)

[0224] Where X1-X 21 As previously defined; Z1 is a cysteine ​​residue or is absent; Z2 is a cysteine ​​residue or is absent. In one embodiment, Z1 is absent and Z2 is a cysteine ​​residue. In an embodiment, at least one of Z1 and Z2 is present. In one embodiment, Z2 is absent and Z1 is a cysteine ​​residue. In one embodiment, Z1 is a cysteine ​​residue and Z2 is a cysteine ​​residue.

[0225] In specific embodiments, the peptide compound has the sequence of GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO:47) or Acetyl-GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO:48) corresponding to SEQ ID NO:10 and SEQ ID NO:15, respectively, modified by adding a cysteine ​​residue at the C-terminus.

[0226] For example, the conjugate compound can comprise 1 to 10 or 1 to 5 (e.g., 1, 2, 3, or 4) molecules of a therapeutic agent (e.g., an anti-tumor agent) attached thereto. These molecules of therapeutic agent 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 at least one covalent bond, at least one atom, or at least one linker. In one embodiment, at least two molecules of the therapeutic agent are attached to A. In one embodiment, the at least two molecules are molecules of the same therapeutic agent (e.g., a chemotherapeutic agent).

[0227] Anti-tumor agents can be any compound that has the ability to inhibit tumor cell growth and / or kill tumor cells, and include, for example, small molecules, peptides, proteins, oligonucleotides (e.g., siRNA, shRNA), radionuclide agents, antibodies, and drug delivery systems, including nanoparticles, liposomes, nanotubes, graphene particles loaded with therapeutic anti-tumor agents.

[0228] In one embodiment, the anti-tumor agent is a chemotherapeutic agent. The term "chemotherapeutic agent" refers to a drug that kills tumor cells and / or inhibits their proliferation / growth. Examples of chemotherapeutic agents include alkylating agents (e.g., cyclophosphamide, ifosfamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, temozolomide, carmustine, lomustine, streptozocin, busulfan, dapoxetine ... usulfan, procarbazine), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), cytoskeletal disruptors (e.g., taxanes, such as paclitaxel, docetaxel, nab-paclitaxel (Abraxane), European paclitaxel (Taxotere), cabazitaxel (ca bazitaxel), 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), efitinib, Imatinib, Vemurafenib, Vismodegib, Dasatinib, Nilotinib, Osimertinib, Crizotinib, Dabrafenib, Vemurafenib, Trametinib, Ibrutinib), nucleotide analogs and precursor analogs (e.g.,Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil (5-FU), Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Tioguanine (Thioguanine), Peptide antibiotics (e.g., Bleomycin, Actinomycin), Platinum-based agents (e.g., Carboplatin, Cisplatin, Oxaliplatin), Retinoids (Tretinoin), , alitretinoin, bexarotene), mitotic inhibitors such as vinca alkaloids and their derivatives (e.g., vinblastine, vincristine, vindesine, vinorelbine), toxins such as maytansinoids, auristatin, calicheamicins, amatoxin or amanitin), and natural phytochemicals with antitumor properties such as curcumin, alkaloids (e.g., chlorogenic acid), acid), Theobromine, Theophylline), Anthocyanins (e.g., Cyanidin, Malvidin), Carotenoids (β-carotene, Lutein, Lycopene), Coumarins, Flavan-3-Ols, Flavonoids (e.g., Epicatechin, Hesperidin, Isorhamnetin, Kaempferol, Myricetin, Naringin, Nobiletin, Proanthocyanidins, Quercetin, Rutin, Tangeretin), 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), Damnacanthal, Digoxin, Phytic acid, Phenolic acids (e.g., Capsaicin, Ellagic acid, Gallic acid, Rosmarinic acid, Tannic acid), Acid), phytosterols (e.g., β-sitosterol), saponins, stilbenes (e.g., pterostilbene, resveratrol), triterpenes (e.g., ursolic acid), xanthophylls (e.g., astaxanthin, β-cryptoxanthin), and monophenols (e.g., hydroxytyrosol).

[0229] In another embodiment, the anti-tumor agent is an antibody or antigen-binding fragment thereof that recognizes an antigen expressed by tumor cells.

[0230] In one embodiment, B is linked to A at the free amine of a lysine residue of the peptide compound, optionally via a linker, or at the N-terminal position of the peptide compound, optionally via a linker. In one embodiment, B is linked to A (optionally via a linker) at a cysteine ​​residue added to the end (e.g., C-terminus) of the peptide compound.

[0231] In one embodiment, B is linked to A via a linker, optionally a cleavable linker.

[0232] As used herein, the term "linker" refers to a chemical structure that connects the peptide compounds disclosed herein to at least one therapeutic agent. The linker can be attached to the peptide compound at different functional groups on the peptide compound. For example, a linker can be attached to a peptide compound at a primary amine (-NH2): This group is present at the N-terminus of each polypeptide chain (called the α-amine) and in the side chain of lysine (Lys, K) residues (called the ε-amine). For example, a linker can be attached to a peptide compound at a carboxyl group (-COOH): This group 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, a linker can be attached to a peptide compound at a sulfhydryl group (-SH): This group is present in the side chain of cysteine ​​(Cys, C). Typically, as part of the secondary or tertiary structure of proteins, cysteines are joined between their side chains by disulfide bonds (-SS-). These species must be reduced to sulfhydryl groups to make them available for cross-linking via most types of reactive groups. For example, a linker can be attached to a peptide compound at a carbonyl group (-CHO): Keto or aldehyde groups can be generated in glycoproteins by oxidizing polysaccharides with sodium metaperiodate for post-translational modification (glycosylation).

[0233] The following table summarizes the reactivity classes and chemical groups of some of the major linkers used in standard chemical conjugates:

[0234]

[0235] For example, homobifunctional and heterobifunctional cross-linkers can be used. For example, disuccinimidyl suberate (DSS) is a homobifunctional cross-linker that has the same amine-reactive NHS ester group at either end of a short spacer arm. For example, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester (sulfo-SMCC) is a heterobifunctional cross-linker that has an amine-reactive sulfo-NHS-ester group at one end of a cyclohexane spacer arm and a sulfhydryl-reactive maleimide group at the other end. This allows for a continuous two-step conjugation procedure. Among the commercially available homobifunctional cross-linkers are: BSOCOES (bis(2\[succinimidyloxycarbonyloxy]ethyl)sulfone; DDPPB (1,4-bis-(3'-[2-pyridyldithio]-propionamide)butane; DSS (disuccinimidyl suberate); DST (disuccinimidyl tartrate); sulfoDST (sulfonamide disuccinimidyl 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 iodide).

[0236] The peptide compound can be conjugated via various linkers (e.g., sulfhydryl, amino (amine) or any suitable reactive group). The linker can be a covalent bond. The linker group can comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms.

[0237] Exemplary linkers include, but are not limited to, pyridyl disulfide, thiosulfonate, vinylsulfonate, isocyanate, imide ester, diazine, hydrazine, thiol, carboxylic acid, polypeptide linker, and acetylene. Alternatively, other linkers that may be used include BS 3 [Bis(sulfosuccinimidyl) suberate] (this is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS / EDC (N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS / EDC allows conjugation of primary amines to carboxyl groups), Sulfo-EMCS ([N-ε-maleimidocaproic acid] hydrazide (Sulfo-EMCS is a heterobifunctional reactive group reactive towards sulfhydryls and amino groups), Hydrazide (most proteins contain exposed carbohydrates, and hydrazides are useful reagents for linking carboxyl groups to primary amines).

[0238] To form a covalent bond, various reactive carboxyl groups (e.g., esters) can be used as chemically reactive groups, wherein the hydroxyl moiety is physiologically acceptable at the level required to modify the peptide compound. Specific agents include, for example, N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimidobenzoyl-succinimide (MBS), γ-maleimidobutyryloxysuccinimide ester (GMBS), maleimidopropionic acid (MPA), maleimidocaproic acid (MHA), and maleimidondecanoic acid (MUA).

[0239] Primary amines are the main targets of NHS esters; NHS esters react with primary amines to form covalent amide bonds. Available α-amine groups present on the N-terminus of proteins and the ε-amine of lysine react with NHS esters. Therefore, the conjugated compounds disclosed herein can include a linker having an NHS ester conjugated to the N-terminal amino group of the peptide compound or to the ε-amine of lysine. When the NHS ester reacts with the primary amine, an amide bond is formed, thereby releasing N-hydroxysuccinimide. Succinimides containing reactive groups can be simply referred to as succinimidyl groups. In some embodiments, the functional group on the peptide compound will be a thiol group, and the chemically reactive group will be a maleimide-containing group, such as γ-maleimido-butyramide (GMBA or MPA). Such maleimide-containing groups may be referred to herein as maleimido groups.

[0240] Amine to amine linkers include NHS esters, imidates, and the like, examples of which are listed below.

[0241]

[0242]

[0243] The linker can also be a thiol-to-thiol linker, such as maleimide and pyridyldithiol listed below.

[0244]

[0245] The linker can be an amine and sulfhydryl linker, including NHS ester / maleimide compounds. Examples of these compounds are provided below.

[0246]

[0247]

[0248] Linkers can react with amino groups and non-selective entities. Such linkers include NHS ester / aryl azide and NHS ester / bis(aziridine) linkers, examples of which are listed below.

[0249] NHS Ester / Aryl Azide Linkers: NHS-ASA (N-hydroxysuccinimidyl-4-azidosalicylic acid) ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide) Sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenzoate) Sulfo-NHS-LC-ASA (Sulfosuccinimidyl[4-azidosalicylamide]hexanoate) SANPAH (N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate) Sulfo-SANPAH (N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate) Sulfo-SFAD (Sulfosuccinimidyl-(perfluoroazidobenzamide)-ethyl-1,3'-dithiopropionate) Sulfo-SAND(Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-propionate) Sulfo-SAED (2-[7-amino-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionic acid sulfosuccinimidyl ester) NHS Ester / Diazirine Linker: SDA (4,4'-azavaleric acid succinimidyl ester) LC-SDA (6-(4,4'-azapentylamido)hexanoic acid succinimidyl ester) SDAD (2-([4,4'-Azapentylamido]ethyl)-1,3'-dithiopropionic acid succinimidyl ester) Sulfo-SDA (4,4'-azapentanoic acid sulfosuccinimidyl ester) Sulfo-LC-SDA (6-(4,4'-azapentylamido)hexanoic acid sulfosuccinimidyl ester) Sulfo-SDAD (2-([4,4'-azapentylamido]ethyl)-1,3'-sulfosuccinimidyl dithiopropionate)

[0250] Exemplary amine and carboxyl linkers include carbodiimide compounds (eg, DCC (N,N-dicyclohexylcarbodiimide) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide)). Exemplary thiol and non-selective linkers include pyridyldithiol / aryl azide compounds (e.g., APDP ((N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide)). Exemplary thiol and carbohydrate linkers include maleimide / hydrazide compounds (e.g., BMPH (N-[β-maleimidopropionic acid] hydrazide), EMCH ([N-ε-maleimidocaproic acid] hydrazide), MPBH 4-(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 and non-selective linkers include hydrazide / aryl azide compounds (e.g., ABH (p-azidobenzoylhydrazide)). Exemplary hydroxyl and sulfhydryl linkers include isocyanate / maleimide compounds (e.g., (N-[p-maleimidophenyl]isocyanate). Exemplary amine and DNA linkers include NHS ester / psoralen compounds (e.g., SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate)).

[0251] To create branch points of varying complexity in the conjugate peptide compounds, linkers can connect 3-7 entities.

[0252]

[0253] TMEA and TSAT can be sulfhydrylated through their maleimide groups. The hydroxyl and carboxyl groups of THPP can react with primary or secondary amines. Other useful linkers conform to the formula Y=C=N–Q–A–C(O)–Z, where Q is a homoaromatic or heteroaromatic ring system; A is a single bond or an unsubstituted or substituted divalent C 1-30 A bridging group, Y is O or S; and Z is Cl, Br, I, N3, N-succinimidyloxy, imidazolyl, 1-benzotriazolyloxy, OAr, wherein Ar is an electron-deficient activated aryl or OC(O)R, wherein R is -A-Q-N=C=Y or a C4-20 tertiary alkyl) (see U.S. Patent No. 4,680,338).

[0254] Other useful linkers are of the formula Where R1 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 6-12 Aryl or aralkyl or with divalent organic -O-, -S- or These groups are coupled, wherein R' is C 1-6 Alkyl, connecting part; R2 is H, C 1-12 Alkyl, C 6-12 Aryl or C 6-12 Aralkyl, R3 is or another chemical structure capable of shifting the lone pair of electrons of the adjacent nitrogen, and R4 is a pendant reactive group capable of linking R3 to the peptide compound (see, eg, US Pat. No. 5,306,809).

[0255] The linker can include 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, the linker can be any naturally or non-naturally occurring amino acid (e.g., Gly or Cys). When the linker is a short peptide, the linker can be a glycine-rich peptide (which is often flexible), such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] n wherein n is an integer from 1 to 6 (inclusive) (see U.S. Patent No. 7,271,149) or a serine-rich peptide linker (see U.S. Patent No. 5,525,491). Serine-rich peptide linkers include those of the formula [XXXX-Gly] ywherein at most two of X are Thr, the remaining X are Ser, and y is an integer greater than 1, such as 1 to 5 (inclusive) (e.g., [Ser-Ser-Ser-Ser-Gly] y (SEQ ID NO: 19), wherein y is an integer equal to or greater than 1, such as 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), wherein n is 1, 2, 3, 4 or 5).

[0256] The linker can be an aliphatic linker (e.g., having an amide bond to the polypeptide and an ester bond to the therapeutic agent). When an aliphatic linker is used, the length of the linker (e.g., C1-C 20 、C1-C 12 , C1-C6) and the included chemical moieties (e.g., amino or carbamate) can vary.

[0257] Examples of suitable amino acid linkers are succinic acid, Lys, Glu and Asp or dipeptides such as Gly-Lys. When the linker is succinic acid, one of the carboxyl groups can form an amide bond with the amino group of the amino acid residue, and the other carboxyl group can, for example, form an amide bond with the amino group of a peptide or a substituent. When the linker is Lys, Glu or Asp, its carboxyl group can form an amide bond with the amino group of the amino acid residue, and its amino group can, for example, form an amide bond with the carboxyl group of the substituent. When Lys is used as a linker, another linker can be inserted between the ε-amino group of Lys and the substituent. Another linker can be succinic acid, which can form an amide bond with the ε-amino group of Lys and with the amino group present in the substituent. In one embodiment, the other linker is Glu or Asp (for example, the other linker forms an amide bond with the ε-amino group of Lys and forms another amide bond with the carboxyl group present in the substituent), that is, the substituent is N-terminated. ε Acylated lysine residues.

[0258] The linker can also be a branched polypeptide. Exemplary branched peptide linkers are described in U.S. Patent No. 6,759,509.

[0259] 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 bound to a linker that reacts with a modified free amine present at a lysine residue in a polypeptide and at the amino terminus of the polypeptide. Thus, the linker for the conjugate compound of the present invention can comprise a group reactive with a primary amine on a polypeptide or a modified polypeptide, to which the therapeutic agent moiety is conjugated. More specifically, the linker can be selected from monofluorocyclooctyne (MFCO), bicyclo\[6.1.0]nonyne (BCN), N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-S-acetylthiopropionate (SATP), maleimide, and dibenzocyclooctyne esters (DBCO esters). In a given linker, useful cyclooctynes ​​include OCT, ALO, MOFO, DIFO, DIBO, BARAC, DIBAC, and DIMAC.

[0260] The linker can comprise flexible arms, such as short arms (<2 carbon chains), medium-sized arms (2-5 carbon chains), or long arms (3-6 carbon chains).

[0261] Click chemistry can also be used for conjugation to peptides (DBCO, TCO, tetrazine, azide, and alkyne linkers). These linker families can be reactive towards amines, carboxyls, and sulfhydryls. In addition, these linkers can be biotinylated, PEGylated, modified with fluorescent imaging dyes, or phosphorylated for incorporation into oligonucleotide sequences.

[0262] In one embodiment, the anti-tumor agent-peptide compound conjugate is represented by formula (LIII) or (LIV):

[0263] GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(LIII) (SEQ ID NO:22);

[0264] Acetyl-GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(LIV) (SEQ ID NO: 23);

[0265] Among them J 1 and J 2 Each is independently an anti-tumor agent (eg, a chemotherapeutic agent) linked to a lysine (K) residue.

[0266] 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 attached 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 attached thereto.

[0267] 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 attached 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 attached thereto.

[0268] In one embodiment, the conjugate compound is GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY - formula (XIX) (SEQ ID NO:28), which comprises a peptide compound having SEQ ID NO:10, wherein each lysine residue has a docetaxel molecule attached thereto.

[0269] 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 attached thereto.

[0270] In one embodiment, 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.

[0271] In another embodiment, the conjugate compound is Acetyl-GVRAK(doxorubicin)AGVRN(Nle)FK(doxorubicin)SESY - Formula (XXVIII) (SEQ ID NO:31), which comprises a peptide compound having SEQ ID NO:15, wherein each lysine residue has a doxorubicin molecule attached thereto.

[0272] In one embodiment, the conjugate compound is GVRAKAGVRN(Nle)FKSESYC(albumin doxorubicin) - formula (LI) (SEQ ID NO:32), which comprises a peptide compound having SEQ ID NO:47, wherein the cysteine ​​residue has an albumin doxorubicin molecule attached thereto, or the conjugate compound comprises a peptide compound having SEQ ID NO:10, wherein a cysteine ​​residue is added to the C-terminus of the peptide compound, and wherein the cysteine ​​residue has an albumin doxorubicin molecule attached thereto.

[0273] In one embodiment, the conjugate compound is acetyl-GVRAKAGVRN(Nle)FKSESYC(albumin doxorubicin) - formula (LII) (SEQ ID NO:33), which comprises a peptide compound having SEQ ID NO:48, wherein the cysteine ​​residue has an albumin doxorubicin molecule attached thereto, or the conjugate compound comprises a peptide compound having SEQ ID NO:15, wherein a cysteine ​​residue is added to the C-terminus of the peptide compound, and wherein the cysteine ​​residue has an albumin doxorubicin molecule attached thereto.

[0274] In one embodiment, the conjugate is administered in the form of a prodrug. As used herein, the term "prodrug" refers to a derivative of the active form of a known compound or composition that is gradually converted into an active form when administered to a subject to produce a better therapeutic response and / or reduced toxicity level. In general, a prodrug will be a functional derivative of the compound disclosed herein that is easily converted into a compound from which it is theoretically derived in vivo. Prodrugs include, but are not limited to, acyl esters, carbonates, phosphates, and polyurethanes. These groups are exemplary, not exhaustive, and those skilled in the art can prepare various other known prodrugs. For example, a prodrug can be formed from available hydroxyl, thiol, amino, or carboxyl groups. For example, the available OH and / or NH2 in the conjugate of the present disclosure can be acylated using an activating acid in the presence of a base and optionally in an inert solvent (e.g., acid chloride in pyridine). Some common esters that have been used as prodrugs are phenyl esters, aliphatic (C1-C 24) esters, acyloxymethyl esters, carbamates, and amino acid esters. In some cases, the prodrugs of the compounds of the present disclosure are prodrugs in which the hydroxyl and / or amino groups in the compound are masked as groups that can be converted into hydroxyl and / or amino groups in vivo. For example, conventional procedures for selecting and preparing suitable prodrugs are described in "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985.

[0275] The covalent modification of conjugate is included in the scope of the present disclosure.Covalent modification includes reacting the targeted amino acid residue of conjugate with an organic derivatizing agent, and the derivatizing agent can react with the selected side chain or N-terminal or C-terminal residue of conjugate.Other modifications include deamination of glutamine and asparagine residues into corresponding glutamine and aspartyl residues, proline and lysine hydroxylation, the phosphorylation of the hydroxyl of seryl or threonyl residues, lysine, arginine and histidine side chains (TECreighton, " Protein: Structure and Molecular Properties (Proteins:Structure and Molecular Properties) ", WH Freeman & Co., San Francisco, 79-86 pages (1983)). Other types of covalent modifications of the conjugates included within the scope of the present disclosure include linking the conjugate to a protein (e.g., albumin) or to a non-protein polymer (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene), which can, for example, increase the in vivo half-life of the conjugate.

[0276] In one embodiment, the conjugate compounds disclosed herein or pharmaceutically acceptable salts thereof are formulated into pharmaceutical compositions. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Such compositions can be prepared by mixing the conjugate compound of appropriate purity with one or more optional pharmaceutically acceptable carriers or excipients in a manner well known in the pharmaceutical art (see Remington: The Science and Practice of Pharmacy, Loyd V Allen, Jr., 2012, 22nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, Rowe et al., 2012, 7th edition, Pharmaceutical Press). The carrier / excipient may be suitable for administration of the conjugate compound by any conventional route of administration, such as oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, or pulmonary (e.g., aerosol) administration. In one embodiment, the carrier / excipient is suitable for administration of the conjugate compound or its salt by intravenous or subcutaneous routes. In one embodiment, the carrier / excipient is suitable for administration of the conjugate compound or its salt by intravenous route. In another embodiment, the carrier / excipient is suitable for administration of the conjugate compound or its salt by subcutaneous route. In another embodiment, the carrier / excipient is suitable for administration of the conjugate compound or its salt by oral route.

[0277] As used herein, " excipient " has the common meaning of this area, and is any composition that itself is not active ingredient (drug).Excipient includes for example adhesive, lubricant, diluent, filler, thickening agent, disintegrant, plasticizer, coating, isolation layer formulation, lubricant, stabilizer, sustained-release agent and other composition.As used herein, " pharmaceutically acceptable excipient " refers to any excipient that does not interfere with the biological activity validity of active ingredient and is nontoxic to experimenter, i.e. is a kind of excipient and / or uses with nontoxic amount to experimenter.Excipient is well known in the art, and system of the present invention is not limited in these respects.In certain embodiments, composition can include excipient, as one or more adhesives (binding agent), thickening agent, surfactant, diluent, sustained-release agent, coloring agent, flavoring, filler, disintegrant / dissolution promoter, lubricant, plasticizer, silicon dioxide flow regulator, glidant, anti-caking agent, anti-adhesion agent, stabilizer, antistatic agent, swelling agent and any combination thereof. As those skilled in the art will recognize, single excipient can bring into play two or more functions simultaneously, for example, both can be used as bonding agent, also can be used as thickening agent.As those skilled in the art will recognize, these terms are not necessarily mutually exclusive.The example of the conventional excipient that is used for injectable formulation comprises water, saline, phosphate buffered saline (PBS), glucose, glycerol, ethanol etc. and its combination.In many cases, in composition, comprise isotonic agent, for example sugar, polyol, as mannitol, sorbitol or sodium chloride will be preferred.The other example of pharmaceutically acceptable material is wetting agent or auxiliary substance, as emulsifying agent, preservative or buffer, and described material increases shelf life or effectiveness.

[0278] The exact amount / dose of the conjugate to be administered will vary depending on various factors, such as the specific cancer cells and the specific cancer disease involved; the extent or degree of involvement or severity of the cancer disease; the size, age and general health of the cancer patient; the response of the individual patient; the specific compound being administered; the bioavailability characteristics of the formulation being administered; the dosage regimen selected; whether the conjugate is administered alone or in combination with other drugs; the pharmacodynamic characteristics of the conjugate and its mode and route of administration; and other relevant characteristics that a physician or a person skilled in the art would readily determine by using known techniques and observing results obtained in similar situations. The conjugate / composition is suitable for administration to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro and then in useful animal models before conducting trials in humans. The present disclosure provides dosages of the conjugate and compositions comprising the conjugate. For example, from about 1 μg / kg to 1000 mg per kilogram (mg / kg) of body weight per day, depending on the type and severity of the disease. In addition, effective dose can be 0.5mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 15mg / kg, 20mg / kg / 25mg / kg, 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg, 50mg / kg, 55mg / kg, 60mg / kg, 70mg / kg, 75mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 125mg / kg, 150mg / kg, 175mg / kg, 200mg / kg, and can be increased progressively by 25mg / kg increments until 1000mg / kg, or its scope can be between any two of the above values. According to the above factors, the scope of typical daily dose can be about 1 μ g / kg to 100mg / kg or more. For repeated administration in several days or longer time, depending on the condition of illness, treatment continues until the expectation suppression of disease symptoms occurs. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0279] The conjugate compounds described herein, or salts thereof, or compositions comprising the conjugate compounds, can be used in combination with one or more additional active agents or therapies (radiotherapy, surgery, vaccines, etc.) for treating target diseases / conditions or for managing one or more symptoms of target diseases / conditions (e.g., analgesics, antiemetics, etc.). In one embodiment, 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 (e.g., colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based drugs, anthracyclines, topoisomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid or its derivatives); geldanamycin or its derivatives (e.g., 17-AAG), and other cancer therapeutics recognized in the art. In some embodiments, the chemotherapeutic agents used in combination with the conjugates described herein include one or more of the following: adriamycin, colchicine, cyclophosphamide, dactinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, stem cell transplantation, or the like. Interferon, camptothecin and its derivatives, cholesterol phenylacetic acid nitrogen mustard, taxanes and their derivatives (for example, taxol, paclitaxel and its derivatives, taxotere and its derivatives, etc.), topotecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ortataxel, gemcitabine, oxaliplatin, Changchunruibin, Capecitabine, Lapatinib, sorafenib, erlotinib, erbitux, derivatives thereof, etc. In one embodiment, the conjugate compound described herein or a composition comprising the conjugate compound is used in combination with an EGFR or tyrosine kinase targeting agent, for example, an EGFR inhibitor (RTK inhibitor). The conjugate compound described herein or a salt thereof or a composition comprising the conjugate compound can also be used in combination with one or more therapeutic antibodies or antibody fragments, for example, therapeutic antibodies or antibody fragments for treating tumors.Examples of antibodies for treating cancer include antibodies targeting CD52 (e.g., Alemtuzumab), VEGF / VEGFR (e.g., Bevacizumab, Ramucirumab), EGFR (e.g., Cetuximab, Necitumumab, Panitumumab), CD38 (e.g., Daratumumab, Isatuximab), RANKL (e.g., For example, Denosumab), GD2 (e.g., Dinutuximab, Naxitamab-gqgk), SLAMF7 (e.g., Elotuzumab), HER2 (e.g., Margetuximab-cmkb, Pertuzumab), CCR4 (e.g., Mogamulizumab), CD20 (Obinutuzumab), Ofatumumab umumab), Rituximab), BCMA (e.g., Teclistamab), CD19 (e.g., Tafasitamab), CTLA-4 (e.g., Tremelimumab), LAG-3 (e.g., Relatlimab), PD-1 (e.g., Tislelizumab, Penpulimab, Sintilimab, Toripalimab), palimab), Retifanlimab, Dostarlimab), PD-L1 (e.g., Durvalumab, Avelumab, Atezolizumab), EpCAM (e.g., Oportuzumab, Edrecolomab), Nephrin-4 (e.g., Enfortumab), CD79b (e.g., Polatuzumab).

[0280] In one embodiment, the conjugate compound defined herein, or a salt thereof, is used in combination with an immunotherapy (eg, an immunotherapeutic agent).

[0281] Therefore, on the other hand, the present disclosure provides a method for treating a subject's cancer, the method comprising administering a therapeutically effective amount of a peptide compound as defined herein or a combination of a conjugate and an immunotherapy (e.g., an immune checkpoint inhibitor therapy) to the subject. The present disclosure also provides the purposes of the peptide compound as defined herein or a conjugate, which is combined with an immunotherapy (e.g., an immune checkpoint inhibitor therapy) for treating cancer or for the preparation of a medicine for treating cancer. The present disclosure also provides a combination therapy for treating cancer, the combination therapy comprising a peptide compound as defined herein or a conjugate and an immunotherapy (e.g., an immune checkpoint inhibitor therapy).

[0282] In one embodiment, the peptide compound or conjugate and the immunotherapy exhibit a synergistic effect in a subject (eg, with respect to inhibition of tumor growth, survival rate, etc.).

[0283] As used herein, the term immunotherapy refers to anti-tumor therapy, which enhances or strengthens the immune response for tumor cells.Immunotherapy includes cell-based immunotherapy, such as administering immune cells that can identify tumor cells, such as chimeric antigen receptor (CAR) T cells and NK cells, or T cells with TCR specific for tumor antigens, or antigen presenting cells (APCs, such as dendritic cells) that can express tumor antigens on their surface.Immunotherapy also includes administering specific antibodies, which recognize the antigens expressed by tumor cells, and targeting the antigens for destruction by the immune system, or administering cytokines (interferons, interleukins) that stimulate immune responses. Another type of immunotherapy includes administering immune checkpoint inhibitors (ICIs). Different types of immunotherapies can be used in combination, such as administering immune cells (CAR T or NK cells) in combination with immune checkpoint inhibitors.

[0284] As used herein, the term "immune checkpoint inhibitor (ICI)" or "immune checkpoint blocker (ICB)" refers to an agent that blocks or inhibits the activity of a negative regulator of an immune response. In one embodiment, ICI blocks or inhibits the activity of T cells (e.g., CTLs and / or CD4 helper T cells) and / or NK cells. Examples of such negative regulators of immune responses (i.e., immune checkpoints) include adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), B and T lymphocyte attenuator (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 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, Penpulimab, Pidilizumab, Sintilimab, Toripalimab, Rivulimab, Dotalimab, Nivolumab, Cemiplimab, Pembrolizumab, Spartalizumab, Camrelizumab, JTX-4014, INC MGA00012 (MGA012), AMP-224, AMP-514), anti-PD-L1 antibodies / blockers (e.g., durvalumab, avelumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189), anti-CTLA-4 antibodies (e.g., tesimumab, ipilimumab), anti-LAG-3 antibodies (e.g., relarilimumab, LAG525 (IMP701), REGN3767 (R3767), BI754,091. Tebotelimab (MGD013), eftilagimod α alpha) (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), Etrumadenant (AB928), Imaradenant (AZD4635), Ciforadenant, NIR178, CS3005, PBF-999, INCB106385, CPI-444), CD73 antagonists / anti-CD73 antibodies (e.g., Mupadolimab (CPI-006), Oleclumab (MEDI9447), Uliledlimab, 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., COM701), anti-CEACAM1 antibodies (e.g., CM24), and CD47 blockers / inhibitors (Evorpacept (ALX148), Hu5F9-G4 (5F9), TTI-662, RRx-001) (see, e.g., Marin-Acevedo et al., Next Generation Immune Checkpoint Inhibitors, and others). Generation of immune checkpoint inhibitors and beyond), Journal of Hematology & Oncology, Vol. 14, Article No. 45 (2021); Xia et al., CD39 / CD73 / A2AR pathway and cancer immunotherapy, Molecular Cancer, Vol. 22, Article No. 44 (2023). The chemical structures and sequences of the above-mentioned immune checkpoint inhibitors are incorporated herein by reference.

[0285] In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1, such as an anti-PD-1 antibody. In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-L1, such as an anti-PD-L1 antibody. In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In one embodiment, a combination of ICIs is used, for example, 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., durvalumab and tesimumab).

[0286] The combination of active agents (e.g., a conjugate compound + an immunotherapeutic agent) and / or a composition comprising the combination can be administered or co-administered (e.g., sequentially, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic agent in a coordinated treatment process to achieve an improved clinical efficacy. Such co-administration may also be performed simultaneously, i.e., during overlapping time periods. 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). In one embodiment, the agents can be combined / formulated into a single composition and thus administered simultaneously.

[0287] In one embodiment, enhancing the anti-tumor immune response comprises (a) increasing the number of tumor infiltrating lymphocytes (TIL), tumor-associated macrophages (TAM) and / or natural killer (NK) cells in the tumor; and / or (b) reducing the level of immune regulatory cells in the tumor. In one embodiment, the TIL comprises activated and / or memory CD4 + and / or CD8 + T cells, such as cytotoxic CD8 + In one embodiment, the TAM comprises type 1 macrophage (M1). In one embodiment, the NK cell comprises a cytotoxic NK cell. In one embodiment, the immune regulatory cell comprises a CD4 + Regulatory T cells (Treg), type 2 macrophages (M2) and / or NK regulatory cells (NKreg).

[0288] Cancer can be any type of cancer, including primary (or original) cancer, recurrent cancer, or metastatic cancer. Examples of cancer include cardiac sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchial cancer (squamous cell adenocarcinoma, undifferentiated small cell adenocarcinoma, undifferentiated large cell adenocarcinoma), alveolar (bronchiolar) cancer, bronchial adenoma, sarcoma (e.g., Ewing's sarcoma, Kaposi's sarcoma, sarcoma), lymphoma, chondromatous hamartoma, mesothelioma, gastrointestinal system cancer, for example, esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach cancer (carcinoma, lymphoma, leiomyosarcoma), gastric cancer (gastric), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma adenocarcinoma, gastrinoma, carcinoid tumor, vipoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colorectal cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract cancer, for example, renal cancer (adenocarcinoma, Wilm's tumor), bladder and / or urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testicular cancer (sperm cell carcinoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); liver cancer ( cancer), for example, hepatoma (hepatocellular carcinoma, HCC), bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vipoma, islet cell tumor and glucagonoma); bone cancer, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostosis), benign enchondroma, chondroblastoma CNS tumors, chondromyoma-like fibromas, osteoid tumors, and giant cell tumors; nervous system cancers, e.g., central nervous system (CNS) tumors, primary CNS lymphomas, skull bone cancers (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges (meningiomas, meningosarcomas, gliomatosis), brain cancers (astrocytomas, medulloblastomas, gliomas, ependymomas, germinal histoma [pinealoma], glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas);Reproductive system cancers, e.g., gynecological cancers, uterine cancer (endometrial cancer), cervical cancer (cervical carcinoma, preneoplastic cervical dysplasia), ovarian cancer (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa cell tumor, Sertoli-Leydig cell tumor, Cancers of the vulva (squamous cell carcinoma, intraepithelial carcinoma, 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; hematologic cancers, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma malignant lymphoma; oral cancer, for example, lip cancer, tongue cancer, gum cancer, palate cancer, oropharyngeal cancer, nasopharyngeal cancer, and paranasal sinus cancer; skin cancer, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, and keloid; adrenal gland cancer: neuroblastoma; and cancers of other tissues, including connective and soft tissue cancer, retroperitoneal and peritoneal cancer, eye cancer, intraocular melanoma and adnexal cancer, breast cancer (for example, ductal breast cancer), head and / or neck cancer (head and neck squamous cell carcinoma), anal cancer, thyroid cancer, parathyroid cancer; secondary and unspecified malignancies of the lymph nodes, secondary malignancies of the respiratory and digestive systems, and secondary malignancies of other sites.

[0289] In one embodiment, the cancer is an immune-cold (unknown) cancer. The term "immune-cold cancer" or "cold cancer" refers to a cancer that does not trigger an anti-tumor immune response in the patient and / or does not respond to cancer immunotherapies such as ICI therapy (see, for example, Bonaventura et al., "Cold tumors: Therapeutic challenges of immunotherapy" Frontiers in Immunology, Vol. 10, 168 (2019). Immune-cold cancers are characterized by the absence of infiltration of anti-tumor immune cells (such as TILs, TAMs and / or NK cells) in the tumor and / or the presence of high levels of immune regulatory cells (e.g., Tregs). Immune-cold tumors can be subdivided into so-called immune desert tumors, in which immune effector cells (such as T cells) are absent from the tumor and its surroundings, and immune-rejection tumors, in which immune effector cells (such as T cells) accumulate near the tumor but cannot effectively infiltrate the tumor. In one embodiment, the immune-cold cancer is an immune desert. In another embodiment, the immune-cold cancer is an immune-rejection tumor.

[0290] Several breast cancers, ovarian cancers, prostate cancers, pancreatic cancers, and glioblastomas are considered immune-cold cancers. Several subtypes of cancer are also considered immune-cold cancers, including subtypes of lung cancer, 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), kidney cancer, such as chromophobe 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). Opportunities, Cancers, Vol. 13, 15, 3652. July 21, 2021), high tumor mutational burden RCC (Yakirevich et al., Tumor mutational burden and immune signatures interplay in renal cell carcinoma. Ann Transl Med 2020; 8(6): 269), colorectal cancer (CRC), such as consensus molecular subtype (CMS) 2 and CMS3 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), esophageal cancer (Puhr et al., Immunotherapy for Esophageal Cancers: What Is Practice Changing in 2021?, Cancer 13, 18, 4632), liver cancer, such as stage II hepatocellular carcinoma (Nguyen et al., Nature Communications 13, Article No.: 1441(2022)). Homozygous deletion of 9p21.3 is one of the most common genomic defects, occurring 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 immunocold phenotype.

[0291] In one embodiment, the methods and uses described herein further comprise the step of determining whether a patient has an immune-cold cancer (or identifying a patient with an immune-cold cancer). Determining whether a patient has an immune-cold cancer can be performed by various methods known in the art. For example, this determination can be performed by evaluating immune cells in the tumor microenvironment (TME), such as TILs (cytotoxic CD8 T cells). + The presence of immune regulatory cells (e.g., Tregs) in the TME indicates that the patient has an immune-cold cancer. By "low number of immune cells" is meant a significantly lower number of immune cells than the average or average number of immune cells detected in a corresponding tumor of the same type; and by "high number of immune regulatory cells" is meant a significantly higher number of immune regulatory cells (e.g., Tregs) than the average or average number of immune regulatory cells detected in a corresponding tumor of the same type.

[0292] In one embodiment, the cancer is resistant to immunotherapy, i.e., a cancer in which immunotherapy does not inhibit tumor growth in the patient. Cancers that are resistant to immunotherapy can be cancers that have never responded to immunotherapy (primary resistance) or cancers that have become resistant to immunotherapy treatment after a period of (responsive) treatment (acquired resistance).

[0293] In other embodiments, the cancer is resistant to therapy based on PD-1 or PD-L1 inhibitors (anti-PD-1 / PD-L1 therapy). In other embodiments, the cancer is resistant to therapy based on PD-1 or PD-L1 inhibitors and is melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer, gastric cancer, squamous cell skin cancer, or myeloma.

[0294] Immune checkpoint inhibitors have been approved or are being tested in Phase III and IV clinical trials for several cancers, including lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer, squamous cell lung cancer), 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 cancer, multiple myeloma, gastric cancer and gastroesophageal junction cancer, gastric adenocarcinoma, melanoma, Merkel cell carcinoma (MCC), lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin 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 (see, e.g., Darvin et al., Experimental & Molecular Medicine). Thus, in one embodiment, the cancer is one of the above cancers for which immune checkpoint inhibitors have been approved or are being tested in Phase III and Phase IV clinical trials.

[0295] Currently approved immune checkpoint inhibitors include anti-CTLA-4 ipilimumab (melanoma and lung cancer), anti-PD-1 nivolumab (melanoma, lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, colon cancer and liver cancer), pembrolizumab (melanoma, lung cancer, head and neck cancer, Hodgkin's lymphoma, renal cell carcinoma and gastric cancer) and cemiplizumab (squamous cell skin cancer, myeloma and lung cancer) and anti-PD-L1 atezolizumab (NSCLC, small cell lung cancer, TNBC) small cell lung cancer, TNBC), avelumab (NSCLC, MCC) and durvalumab (urothelial carcinoma, lung cancer). Therefore, in one embodiment, the cancer is one of the above cancers for which immune checkpoint inhibitors have been approved.

[0296] In one embodiment, the dose of the conjugate compound described herein and / or immunotherapy used / administered in the methods, uses, compositions, and combination therapies of the present disclosure is a suboptimal dose. As used herein, a "suboptimal dose" refers to a dose of one of the compounds in the combination described herein (peptide compounds and / or immunotherapy described herein) that, when used in the absence of another compound in the combination, produces a biological effect of 50% or less, in one embodiment 40% or less, in another embodiment 30% or less, in another embodiment 20% or less, in another embodiment 10% or less. Thus, using a combination of the compounds described herein in which one or more compounds in the combination are used at a suboptimal dose can achieve increased therapeutic efficacy / biological effect compared to using the compound at a comparable suboptimal dose in the absence of the other compound.

[0297] As described herein, a synergistic effect occurs when the effect of the combined compounds is greater than the theoretical sum of the effects of each agent in the absence of the other. A potential advantage of combination therapies with synergistic effects is that lower doses (e.g., suboptimal doses) of one or both of the drugs or therapies can be used to achieve high therapeutic activity and low toxicity. In one embodiment, the effect of the combination therapy (peptide compounds and / or immunotherapy described herein) is increased by at least 5% relative to the predicted theoretical additive effect of the agents. In another embodiment, the effect of the combination therapy is increased by at least 10% relative to the predicted theoretical additive effect of the agents. In another embodiment, the effect of the combination therapy is increased by at least 20% relative to the predicted theoretical additive effect of the agents. In another embodiment, the effect of the combination therapy is increased by at least 30% relative to the predicted theoretical additive effect of the agents. In another embodiment, the effect of the combination therapy is increased by at least 50% relative to the predicted theoretical additive effect of the agents. Another advantage of using combined drugs is that therapeutic effects that would not be achieved if either drug were used alone can be achieved, such as for cancers or tumors that are resistant to ICIs. Drug resistance means that the administration of ICI alone does not produce a significant therapeutic effect, for example, a significant reduction in tumor volume or number of tumor cells or an extension of survival time. Examples of cancers that have been reported to be resistant to ICI in patients and / or animal models include lung cancer (e.g., NSCLC), pancreatic cancer, prostate cancer, melanoma, ovarian cancer, urothelial carcinoma, renal cell carcinoma (see, for example, 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 one embodiment, the synergistic effect is to inhibit or reduce tumor growth. In one embodiment, the synergistic effect is to increase survival time.

[0298] Therefore, in other aspects, the present disclosure also provides a kind of combination therapy, which includes peptide compounds as described herein and immunotherapy, such as immune checkpoint inhibitors (ICI) therapy. The present disclosure also provides a kind of combination therapy purposes, the combination therapy includes peptide compounds as described herein and immunotherapy (that is, immunotherapeutic agent), such as ICI, which is used to treat the subject with cancer (for example, to immunotherapy, such as ICI monotherapy with drug-resistant cancer). The present disclosure also provides a kind of combination therapy purposes, the combination therapy includes peptide compounds as described herein and immunotherapy (such as immune checkpoint inhibitors), the peptide compounds and immunotherapy are used to prepare medicines, and the medicines are used to treat the subject with cancer (for example, to immunotherapy, such as ICI monotherapy with drug-resistant cancer). The present disclosure also provides a kind of method for treating the subject with cancer (for example, to immunotherapy, such as ICI monotherapy with drug-resistant cancer), the method including administering an effective amount of combination therapy to the subject, the combination therapy including peptide compounds as described herein and immunotherapy (such as ICI).

[0299] As used herein, the term "subject" or "patient" refers to mammals, such as rodents, felines, canines, and primates. The subject or patient according to the present disclosure is preferably a human.

[0300] Examples

[0301] The present invention is further illustrated by the following non-limiting examples.

[0302] Example 1: Materials and Methods

[0303] Cells and reagents.

[0304] Human SK-MEL-28 melanoma cells with stage I and stage II melanosomes were obtained from the American Type Culture Collection (ATCC, Manassas, VA; No. HTB-72) [17, 18] and cultured in Eagle's Minimum Essential Medium (EMEM; Wisent, No. 217-010-XK) containing 1 mM sodium pyruvate and 10% fetal bovine serum (FBS; Hyclone, No. SH30396.03) at 37°C in a humidified atmosphere (5% CO2). Human A375 melanoma cells were also obtained from ATCC (No. CRL-1619) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; No. 319-005-CL) containing 10% FBS. Human TNBC-derived MDA-MB-231 / Luc cells were obtained from CellBiolabs, Inc. (San Diego, CA; No. AKR-231) and cultured in DMEM containing 10% FBS. Stage III / IV melanosome model [19, 20] murine B16-F10 melanoma cells (ATCC; No. CRL-6475) were cultured in DMEM with 10% FBS at 37°C in a humidified atmosphere (5% CO2) and used for in vivo syngeneic studies. Cell counts and cell viability were assessed using a BioRad TC20 automated cell counter. A polyclonal anti-SORT1 antibody directed against amino acid 800 to 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 (No. 124312) and APC rat immunoglobulin G (IgG) 2b, kappa isotype control antibody (No. 400612) were obtained from Biogen (San Diego, CA). APC major histocompatibility complex class I (MHC-1; H-2Db) monoclonal antibody (mAb; 28-14-8; No. 17-5999-82) and APC mouse IgG2a kappa isotype control (eBM2a) (No. 17-4724-81) were obtained from Thermo Fisher Scientific (Agawam, MA). All other reagents were from Sigma-Aldrich (Oakville, ON).

[0305] Tissue microarray probing and analysis.

[0306] SORT1 expression was evaluated at the Institute of Immunology and Cancer Research (IRIC; Montreal, QC) using high-density tissue microarrays (TMA) of human melanoma cancer and healthy tissue. TMA provides isoform information, allowing the assessment 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 cells were stained with diaminobenzidine chromogen using Bond Immunofluorescence ... TM A polymer refinement assay (Leica Biosystems, Buffalo Grove, IL) detected the target antigen for visualization. Sections were then counterstained with Leica's proprietary hematoxylin and mounted for analysis. Images from immunohistochemistry were analyzed by a 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; 3, strong staining. Raw data were converted to IHS by multiplying the amount by the staining intensity score. Therefore, the IHS score ranges from 0 to 12.

[0307] Western blotting

[0308] The cells were homogenized in 1% sodium dodecyl sulfate (SDS) lysis buffer supplemented with complete protease inhibitor cocktail from Calbiochem (San Diego, CA). The cells were incubated for 30 minutes at room temperature (RT), vortexed every five minutes, sonicated, and centrifuged at 15,000 g for 10 minutes at room temperature. Equal amounts of protein (20 μg) were separated by SDS polyacrylamide gel electrophoresis (PAGE). The proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes and eluted with 0.1% Tween-40 at room temperature. TMThe membranes were blocked for one hour with 5% nonfat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.5) in Tris-buffered saline (TBST). The membranes were washed in TBST and incubated overnight with primary antibodies against SORT1 (1 / 1,000 dilution) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAb 6C5, 1 / 40,000 dilution) diluted in TBST containing 3% bovine serum albumin (BSA) and 0.05% NaN3. STING (No. 50494, 1 / 1,000 dilution), p21 (No. ab109199, 1 / 1,000 dilution), TANK-binding kinase 1 (TBK1; No. 3504, 1 / 1,000 dilution), phospho-TBK1 (No. 5483, 1 / 1,000 dilution), P65 (No. 8242, 1 / 1,000 dilution), phospho-P65 (No. 3033, 1 / 1,000 dilution), and p53 (No. ab131442, 1 / 5,000 dilution). The membrane was washed in 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 one hour at room temperature. The membrane was washed again in TBST, and the signal was detected using chemiluminescence (Bio-Rad, Saint-Laurent, Quebec).

[0309] animal

[0310] Female CD-1 nude mice (Crl:NU-Foxn1 nu , 4-6 weeks old) are used for xenograft tumor model, and female immunocompetent C57BL / 6 mice (C57BL / 6NCrl, 4-6 weeks old) are used for syngeneic tumor model.All mice are obtained from Charles River Laboratories, Inc. (St-Constant, Quebec).Animals are adapted for 5 days before the experiment.All mice are maintained in a pathogen-free environment and are handled according to the laboratory animal care and use guidelines of the Canadian Council on Animal Care (CCAC).

[0311] Preparation of test articles for injection

[0312] TH1902 (Acetyl-GVRAK(Docetaxel)AGVRN(Nle)FK(Docetaxel)SESY) was synthesized as previously described (PCT Publication No. WO 2017 / 088058). A stock solution of formulated TH1902 (10 mg / ml) was prepared as sterile aliquots and frozen. On the day of animal dosing, the frozen aliquots were thawed and then diluted with sterile 5% dextrose injection USP (D5W) to the desired injection concentration. The frozen stock solution of the vehicle was processed exactly according to the dilution of the TH1902 stock solution to match the amount of excipient present in the TH1902 animal group. Docetaxel (Wonda Science Inc, Lexington, MA) was prepared on the same day as the animals were dosed and matched the docetaxel content in the highest administered TH1902 dose for comparison. Docetaxel is dissolved to 50 mg / ml with EtOH for injection, diluted to 25 mg / ml with polysorbate-80, and then diluted to the required injection concentration (i.e., 2.5 mg / ml) with sterile 5% D5W. All diluted solutions are filtered (Millex-GP 0.22 μm syringe filter, PES membrane, Millipore) before animal administration. It is worth noting that, in terms of docetaxel content, a 15 mg / kg docetaxel dose is equivalent to 35 mg / kg TH1902. Similarly, a 7.5 mg / kg docetaxel dose is equivalent to 17.5 mg / kg TH1902 (the conversion factor of 2.3 takes into account the molecular weight of two docetaxel parts 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 injection concentration 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 administration to animals.

[0313] In vivo efficacy evaluation of docetaxel and TH1902 in the MDA-MB-231 immunocompromised xenograft model and the B16-F10 syngeneic immunocompetent melanoma model.

[0314] The MDA-MB-231 xenograft model has been described previously [4,5]. The B16-F10 melanoma syngeneic model was generated as follows: B16-F10 cells were resuspended in 100 μl of implantation medium (Millipore Sigma; HBSS, No. H6648) and Matrigel (Corning Inc., Corning, NY; No. 356231) and injected at a 1:1 ratio of 1×10 in 100 μl. 5 cells (1x10 6 Cells / ml). Under mild isoflurane anesthesia, tumors were established by subcutaneously inoculating B16-F10 cells into the dorsal region of immunocompetent C57BL / 6 mice. The injection schedule, dosage, number of treatment cycles, and method of administration of the injected substance are described in detail in the accompanying drawings. Briefly, mice were treated weekly with vehicle, docetaxel (15 mg / kg), or TH1902 (35 mg / kg) via intravenous (IV) tail vein injection. When the average size of the MDA-MB-231 tumor reached approximately 100 mm 3 Or three days after implantation of B16-F10 cells, treatment was started. MDA-MB-231 tumors were collected under the following conditions: (i) four days after treatment with vehicle, docetaxel or TH1902 for three cycles; (ii) four days after treatment with TH1902 for six cycles; or (iii) four days after treatment with TH1902 for six cycles, and then three cycles of treatment were stopped to observe tumor growth over the long term. B16-F10 tumors were collected four days after treatment with vehicle, docetaxel or TH1902 for two cycles. Tumors were collected at their respective time points and then fixed in 10% buffered formalin and subjected to immunohistochemistry (IHC) analysis. For all in vivo studies, tumor growth was monitored by two-dimensional measurements collected with electronic calipers, and tumor volume was calculated by the following formula: tumor volume (mm 3 )=π / 6xlengthxwidth 2 Animal body weight was measured with an accuracy of ±10 mg.

[0315] In vivo evaluation of the combined efficacy of docetaxel or TH1902 with anti-PD-L1 antibody in the B16-F10 syngeneic model.

[0316] The generation of B16-F10 melanoma isogenic model is carried out as described above. The first study investigated the effect of docetaxel, TH1902 or anti-PD-L1 alone or in combination (docetaxel / anti-PD-L1 or TH1902 / anti-PD-L1) on the tumor growth of primary tumors three days after B16-F10 cells were implanted into mice, and the mice were treated for two cycles. The treatment dose and schedule (indicated in the legend) for each cycle are as follows: (i) a control group (9 mg / kg, intraperitoneal, once every two weeks) treated with an appropriate vehicle and an isotype control of anti-PD-L1; (ii) half of its maximum tolerated dose of docetaxel (MTD; 7.5 mg / kg, intravenous, once a week); (iii) an equivalent dose of TH1902 (17.5 mg / kg, intravenous, once a week) of docetaxel; (iv) a combination of docetaxel and anti-PD-L1; (v) a combination of TH1902 and anti-PD-L1. The second study investigated the effects of increasing doses of TH1902 (4.37, 8.75, and 17.5 mg / kg, intravenously, weekly) or anti-PD-L1 (9 mg / kg, intraperitoneally [IP], biweekly) alone or in combination on mouse survival. Treatment cycles continued until one of the study endpoints (tumor size >2,000 mm) was achieved. 3 , weight loss >20% of initial body weight, tumor ulceration, death). In both studies, tumor growth and mouse body weight were monitored as described above.

[0317] In vivo efficacy evaluation of docetaxel and TH1902 using a syngeneic Lewis lung carcinoma (LL / 2) xenograft model.

[0318] 1x10 cells were inoculated subcutaneously in 100 μl (LL / 2) HBSS / basement membrane (50:50). 6 The cells were used to establish tumors. Under mild isoflurane anesthesia, all cells were injected into the dorsal region of immunocompetent (C57BL / 6) mice. For LL / 2 tumors, mice were treated with vehicle, docetaxel (15 mg / kg) or TH1902 (35 mg / kg) of an equivalent docetaxel amount via intravenous (IV) tail vein injection every week for 3 days after implantation until 11 days, covering 2 treatments (0th day and 7th day after starting treatment). For all specified studies, tumor growth was monitored by two-dimensional measurements collected with an electronic caliper, and tumor volume was calculated by the following formula: tumor volume (mm 3 )=π / 6xlengthxwidth 2 Animal body weights were measured with an accuracy of ±10 mg. When the tumor size of mice in the vehicle group reached approximately 600-1000 mm 3 Tumors were collected at 4 hr.

[0319] Immunohistochemical staining was performed on tissue microarrays and in vivo tumors.

[0320] SORT1 expression was evaluated at the Institute of Immunology and Cancer Research (IRIC; Montreal, Quebec) using high-density tissue microarrays (TMAs) of human melanoma (IMH-366; Novus Biologicals, Centennial, CO) and healthy tissue (IMH-373; Novus Biologicals). TMAs provide subtype information, allowing the assessment of SORT1 expression in melanoma samples of different grades. Immunostaining of TMAs was performed as previously described [5]. IHC images were analyzed by pathologists. SORT1 labeling was scored using the IHS method on a scale of 0 to 3: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. Raw data were converted to IHS scores by multiplying the amount by the staining intensity score. Thus, the IHS score ranges from 0 to 12.

[0321] After sacrifice, primary MDA-MB-231 and B16-F10 tumors were isolated from mice, fixed in 10% buffered formalin, and then embedded in paraffin. Hematoxylin and eosin (H&E; 23-314631 and 23-245657, Fisher Scientific) staining was performed to evaluate gross morphology. The Leica Bond Max automated platform (Leica Biosystems, Nussloch) with the bond polymer thinning detection kit (DS9800, Leica Biosystems) was used. GmbH)) performed IHC on serial slides. Briefly, 4-micron sections were collected from tissue blocks on coated slides. The sections were then deparaffinized, hydrated, and blocked with hydrogen peroxide. Appropriate buffers were used for each antibody to achieve heat-induced or enzymatic antigen retrieval. 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 incubation with secondary antibodies. Staining was accomplished with diaminobenzidine (DAB) chromogen and hematoxylin was used as a counterstain. Images were acquired using National Institutes of Health (NIH) ImageJ. Quantification of immune cells infiltrating tumor nuclei was performed using software version 1.4.21. The mean color intensity for each staining, using the same threshold setting, was divided by the tumor area of ​​interest and expressed as a percentage of the positive area. For each section, the entire tumor surface was analyzed, excluding the tumor margin, invasive margin, and necrotic areas. Table 1 provides detailed information on all primary antibodies, commercial suppliers, dilutions, antigen retrieval, and processing conditions.

[0322] Table 1: List of antibodies and staining conditions used in the immunohistochemistry experiments described herein

[0323]

[0324]

[0325] 1 Primary antibody dilutions were with or without blocking solution (Bk; Leica Biosystems, No. PV6122).

[0326] 2 H1: HIER 1, citrate buffer, pH 6, Leica Biosystems, catalog number AR9961; HIER 2, EDTA buffer, pH 9, Leica Biosystems, catalog number AR9640; ENZ: enzymatic digestion, Leica Biosystems, catalog number AR9551.

[0327] 3 Incubate primary antibody / secondary antibody / polymer.

[0328] 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. The sections were then treated with 1% periodic acid-Schiff (PAS) solution for 10 minutes. After rinsing with distilled water for two minutes, the tumor tissue sections were placed in Schiff solution in a darkroom for 30 minutes and rinsed three times with distilled water. Finally, the slides were counterstained with Leica proprietary hematoxylin and mounted for analysis. Healthy blood vessels were CD31-positive / PAS-positive, while VM-associated structures were CD31-negative / PAS-positive. Images were captured using a Nanozoomer slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) and analyzed using an Aperio ImageScope (Leica Biosystems, Buffalo Grove, IL, version 12.4.3.5008).

[0329] Cell proliferation assay.

[0330] To evaluate the effects of docetaxel and TH1902 on the proliferation of SK-MEL-28 and B16-F10 melanoma cells, cells were seeded in 96-well plates (PerkinElmer, Waltham, MA) and treated with different concentrations of drugs in complete cell culture medium. After incubation for 120 h 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 four hours at 37°C in a humidified atmosphere containing 5% CO2. After incubation, 100 μL of DMSO (solubilizing agent) was added to each well and mixed thoroughly for five minutes to dissolve the dark blue crystals. The presence of living cells was visualized by the purple coloration due to the formation of formazan crystals. A test wavelength of 570 nm and a reference wavelength of 650 nm were used in the SpectraMax TM Plates were read on a BIOMEDIA® Plus plate reader (Molecular Devices, San Jose, CA). Analyses were performed in quadruplicate in each case.

[0331] Apoptosis assay.

[0332] Annexin V / PI staining was performed using an apoptosis detection kit (BD Pharmingen, San Diego, California) according to the manufacturer's instructions. Briefly, B16-F10 cells were treated in serum-free medium containing docetaxel (0.1 or 2 μM) or TH1902 (0.05 or 1 μM) for 15 minutes. The cells were then washed twice with complete growth medium and incubated for 94 hours in complete growth medium. The cells were harvested and resuspended in a staining solution of 100 μL of 1x binding buffer containing 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI). Finally, the cells were incubated in the dark at room temperature for 15 minutes; a BD AccuriC6 flow cytometer (BD Biosciences, Franklin Lakes, NJ) was used to collect and analyze the number of apoptotic cells.

[0333] Senescence activity assay.

[0334] B16-F10 cells were plated at a density of 20,000 cells / well in a 1% 4% PBS (ibidi, Grafelfen, Germany). After 24 hours, the cells were treated with serum-free medium containing 7.5 μM docetaxel or 3.75 μM TH1902 for two hours. The cells were then washed twice with complete growth medium and incubated in complete growth medium for four days. As a positive control for senescence, the cells were treated with serum-free medium containing 12.5 μM etoposide for 24 hours, washed, and incubated in complete growth medium for up to four days. CellEvent was used according to the manufacturer's instructions. TM Senescence Green Detection Kit (Thermo Fisher Scientific, No. C10850) was used to detect senescence-associated β-galactosidase (SA-β-gal) activity. Micrographs were captured using a confocal microscope (Nikon A1plus, Melville, NY) and digitized at 20x magnification, then analyzed using NIH ImageJ version 1.4.21 software. For cell morphology assessment, cells were fixed with 10% formalin phosphate, stained with 0.1% crystal violet / 20% methanol, and observed microscopically.

[0335] Total RNA isolation, cDNA synthesis, and real-time quantitative PCR.

[0336] Qiagen RNeasy TMTotal RNA was extracted from the cell monolayer using a QIAgen kit (Qiagen, Toronto, Ontario). 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). PCR products were analyzed using SsoFast EvaGreen Supermix. TM (1725201, Bio-Rad, Hercules, CA) quantified gene expression by real-time quantitative PCR. 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 α (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. The amplification curve (fluorescence signal relative to the number of cycles) was followed by the ΔC T Methods RNA was measured. The difference (ΔC) between the mean values ​​of triplicate samples of target genes and housekeeping genes was calculated using CFX Manager software version 2.1 (Bio-Rad). T ), and the relative quantification value (RQV) was expressed as 2 -ΔCT .

[0337] Flow cytometry.

[0338] A single B16-F10 live cell suspension was washed with RPMI-1640, supplemented with 8% FBS in PBS, and washed once with FACS buffer. Subsequently, the sample was incubated with Fc blocking mice (2%) and mouse serum (5%) for 10 minutes, then stained with anti-MHC-I or anti-PD-L1 antibodies for one hour at 4 ° C in the dark, and finally rinsed twice with PBS containing 0.5% BSA solution. Samples were collected using LSR Fortessa (BD Biosciences) and the results were analyzed using FlowJo software.

[0339] Statistical analysis

[0340] Data are presented as mean ± standard error of the mean (SEM) or standard deviation (SD) as indicated in the figure legends. Statistical analysis was performed using the t-test for comparisons of two samples, while analysis of three or more samples was performed by one-way ANOVA followed by Bonferroni's multiple comparison, Dunnett's multiple comparison, or Tukey's multiple comparison. Values ​​of p < 0.05 (*) and p < 0.01 (**) were considered significant, and asterisks (*) indicate these levels of significance in the figures.

[0341] Example 2: Sustained and prolonged antitumor activity of TH1902 in the immunosuppressive MDA-MB-231 TNBC-derived xenograft model is associated with increased immune cell infiltration and STING expression.

[0342] First, the efficacy of TH1902 and docetaxel for TNBC xenograft models was studied in vivo. Immunosuppressed nude 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 (i) docetaxel (three treatments) or (ii) TH1902 (six treatments) with an MTD of 15 mg / kg / wk intravenous bolus injection at 35 mg / kg / wk. The other group received TH1902 (iii) six treatments and was monitored until day 60 ( Figure 1A ). Treated tumors were collected after treatment was stopped, and in the case of TH1902, tumors were collected on day 60. Compared with docetaxel, TH1902 only halved tumor growth at an equivalent dose, but TH1902 induced complete tumor regression and maintained this effect over the six treatments until day 60 (i.e., 25 days after the last treatment). At the same time, there was almost no change in body weight ( Figure 1E H&E staining of the resected tumors showed that the morphology of SORT1-positive cancer cells was significantly enlarged after six cycles of treatment; this morphology remained unchanged after the end of treatment ( Figure 1B ). IHC analysis further revealed that Ki67 cell proliferation was reduced and STING and CD45 staining were increased in TH1902-treated tumors, and SORT1 was continuously expressed on cancer cells after treatment ( Figure 1C After treatment, increased CD45 immunoinfiltration staining specifically surrounded the enlarged cancer cells ( Figure 1C ). The anti-angiogenic mimetic (VM) properties of TH1902 were observed as early as the third cycle of treatment, such that CD31 - / PAS + Reduced staining ( Figure 1D In contrast, docetaxel appears to have much less effect on Ki67, STING, and CD45 staining, as well as on VM, than TH1902. Interestingly, unlike docetaxel, TH1902 significantly induced the expression of both p21 and p53, which are associated with cellular senescence, in MDA-MB-231 cells in vitro ( Figure 1F -G). Finally, Figure 1H -The results depicted in Figure 1 show that treatment with TH1902 increases PD-L1 expression in MDA-MB-231 tumors in vivo. Taken together, these results demonstrate that TH1902 has the ability to alter the immune tumor microenvironment (iTME) through the process of immune cell infiltration and is associated with a durable effect on tumor regression.

[0343] Example 3: SORT1 is highly expressed in clinically annotated melanoma tissue and cell line models.

[0344] To evaluate whether TH1902 could act as a chemotherapeutic pathway mediated by the SORT1 receptor in immunocompetent syngeneic models, SORT1 expression was assessed in murine B16-F10 melanoma cells and other cell lines. This screen was also performed in healthy skin tissue and TMA of clinically annotated stage II-IV melanoma tumors by IHC ( Figure 2A IHS scores showed that SORT1 expression increased throughout these stages, while levels remained low in healthy tissues ( Figure 2B High SORT1 expression was also verified in lysates from the murine B16-F10 melanoma cell line, as well as from two human melanoma cell lines (A375 and SK-MEL-28), and from a positive control (human TNBC-derived MDA-MB-231). Figure 2C The high expression of SORT1 in murine B16-F10 cells validates this syngeneic model for evaluating the in vivo therapeutic effect of TH1902 on iTME.

[0345] Example 4: TH1902 exhibits anti-proliferative and pro-apoptotic activities in vitro and induces cancer cell senescence.

[0346] Select SORT1 positive SK-MEL-28 ( Figure 3A ) and B16-F10( Figure 3B ) melanoma cells to test the antiproliferative effects of docetaxel and TH1902. When monitoring the biological effects of TH1902, the half-maximal inhibitory concentration (IC 50 ) are similar to the half-maximal inhibitory concentrations of docetaxel, which are on average 0.38 and 0.39 nM in human SK-MEL-28 cells, and on average 2.57 and 1.72 nM in murine B16-F10 cells, respectively ( Figure 3C ), indicating that the antiproliferative properties of docetaxel are not affected after it is conjugated to the cleavable linker of TH19P01. Since the cytotoxicity of docetaxel is related to its ability to induce cell cycle arrest and apoptosis, the apoptotic effects of low and high concentrations of docetaxel and TH1902 on B16-F10 cells were studied. The cells were treated with 0.1 or 2 μM docetaxel ( Figure 3D , black bars) or 0.05 and 1 μM TH1902 (carrying equimolar conjugated docetaxel concentrations; Figure 3D , gray bars) for 15 minutes, rinsed, and further incubated in complete medium for up to 96 hours. Apoptosis was then assessed by flow cytometry as described in Example 1. Although only high docetaxel concentrations induced apoptosis, TH1902 induced progressively higher apoptosis in a dose-dependent manner ( Figure 3D Given that docetaxel is known to induce senescence in B16-F10 cells

[22] and limit proliferation capacity

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

[0347] Example 5: TH1902 blocks tumor growth and triggers leukocyte infiltration in an immunocompetent syngeneic “cold” tumor model.

[0348] Generation of B16-F10 melanoma syngeneic tumors ( Figure 4A ), and tumor size was monitored as described in Example 1. Tumors in vehicle-treated mice bearing xenografts grew exponentially ( Figure 4A , black circles). After intravenous administration of 15 mg / kg / wk docetaxel, partial inhibition of tumor growth was observed ( Figure 4A , squares), whereas treatment with docetaxel-equivalent amounts of TH1902 (35 mg / kg / wk) induced tumor regression after two treatments within the measured period ( Figure 4A, triangles). Due to rapid tumor growth, only two administrations of the test article could be performed on a weekly schedule. B16-F10 melanomas from mice treated with vehicle, docetaxel, or TH1902 were then excised, fixed in formalin, and subjected to immunohistochemistry ( Figure 4B Within the tumor shown in the figure, necrotic areas were present, and some areas were filled with actual cancer cells with significantly enlarged morphology, as previously observed in TH1902-treated MDA-MB-231 tumors ( Figure 4C Here too, a large infiltration of immune cells (CD45+ total leukocytes) was detected in tumors from TH1902-treated animals ( Figure 4D Tumor parenchyma from vehicle-treated animals showed little immunodetection of CD45 ( Figure 4C , left panel), thereby indicating the absence of leukocytes, whereas tumors from animals treated with docetaxel showed only slightly higher leukocyte infiltration ( Figure 4C , middle panel). However, in both cases, staining was restricted to the tumor periphery. This suggests that the tumors have an immune exclusion phenotype, rather than the related immune desert phenotype, in which T cells are clearly absent from the tumor parenchyma or stroma

[24] . It goes without saying that tumors lacking lymphocyte infiltration are less likely to respond to checkpoint inhibitors (CPIs); this has been established with respect to lymphocyte infiltration [25,26]. However, animals treated with TH1902 showed significantly higher tumor levels of staining for the pan-immune cell marker CD45 ( Figure 4C , right panel), and quantitatively confirmed that most leukocytes infiltrated within the tumor parenchyma ( Figure 4D As a rough indicator of morbidity, the body weight of mice treated with the test article or vehicle was tracked. Mice bearing B16-F10 tumors showed similar body weights ( Figure 4E ), indicating that the animal's weight is still within the acceptable range.

[0349] Example 6: TH1902 triggers immune cell infiltration in tumors.

[0350] The immune response to tumors is a complex and coordinated process involving numerous cell types, interacting membrane proteins, and soluble effectors

[27] . By IHC, docetaxel induced a moderate infiltration of all subclasses of lymphocytes, including cytotoxic T cells, helper T cells, regulatory T cells, and natural killer (NK) cells within the tumor parenchyma ( Figure 5A , horizontal middle panels). However, the increase produced by TH1902 treatment was systematically and significantly greater than that produced by docetaxel treatment in all lymphocyte classes measured ( Figure 5A , horizontal bottom panel) and Figure 5B , gray bars). Compared to the vehicle, CD3 T cell markers were slightly increased with docetaxel and significantly increased with TH1902. IHC data also showed that TH1902 treatment significantly increased the expression of CD8+ (or cytotoxic) T cells, which are known to have anti-tumor immune responses. In addition, TH1902 increased the infiltration of CD4 T cells more than docetaxel, while TH1902 induced the level of regulatory T cell (Treg; FoxP3) infiltration. In addition, the expression of CD161c NK markers was increased in TH1902-treated mice relative to mice treated with vehicle or docetaxel ( Figure 5B ).

[0351] Macrophages also represent an important class of immune cells within tumors. In fact, tumor-associated macrophages (TAMs) are often the most abundant immune cell population within most tumors

[28] . TAMs can enhance or antagonize the cytotoxic activity of immune cells, and their effects are usually attributed to two different subsets, M1 and M2

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

[30] . Similar to tumor infiltrating lymphocytes (TILs), tumor-infiltrating TAMs (F4 / 80 + ) and the number of M1(CD68 + ) and M2(CD206 + ) The number of macrophages was slightly increased by docetaxel treatment but significantly increased by TH1902 treatment ( Figure 5C and 5D ). Surprisingly, for TH1902, CD206 + The expression of M2 macrophage markers showed a correlation with CD68 + The same trend was observed for M1 macrophages. Thus, stimulation of leukocyte infiltration of tumors by TH1902 administration is applicable to multiple classes of immune cells.

[0352] Example 7: TH1902 induces apoptosis in response to immune stimulation.

[0353] Regardless of the specific lineage of cells infiltrating the tumor, 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 through the granzyme B / perforin apoptotic pathway. Therefore, the effects of 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 6AHowever, after treatment with TH1902, there was a significant increase in perforin, granzyme B, and caspase-3 staining (5- to 7-fold increase compared to docetaxel), suggesting that TH1902-induced tumor cell apoptosis is at least partially mediated by immune cells ( Figure 6B Together, these data support a clear effect of TH1902 on the anti-tumor response of CD8+ T cell effectors and NK cells, which resulted in complete tumor regression following cancer cell death.

[0354] Example 8: Effect of TH1902 / anti-PD-L1 combination on B16-F10 tumors.

[0355] Treatment of B16-F10 tumors with TH1902 appears to allow extensive infiltration of the tumor by immune cells and greatly enhances apoptosis induced by immune cells. TH1902 also appears to circumvent the immune rejection properties of these “cold” tumors. Therefore, combining TH1902 with a checkpoint inhibitor (CPI) appears feasible. Since anti-PD-L1 immunotherapy is known to be ineffective in the B16-F10 syngeneic model

[32] , the combination of this antibody with docetaxel or TH1902 was evaluated. To evaluate the synergistic therapeutic effect, the doses of docetaxel and TH1902 were halved. As observed, tumor growth was similar in the vehicle group and in mice treated with an isotype control antibody (anti-PD-L1 control), while administration of anti-PD-L1 itself produced a small and non-statistically significant 35% reduction in tumor growth (p=0.0702). These results are similar to those previously reported for this tumor model

[33] , where docetaxel administration produced a significant but small 49% reduction in tumor growth, while TH1902 produced a larger, significant 92% reduction ( Figure 7A ). Treatment with the combination of anti-PD-L1 and docetaxel produced a further small decrease in tumor growth (66%), while the combination of anti-PD-L1 and TH1902 significantly reduced tumor size, showing tumor regression on day 11 (30% regression compared to the initial tumor volume at the start of treatment). As the experiment continued, the enhanced inhibition of tumor growth by the combination of TH1902 and anti-PD-L1 became increasingly apparent. Monotherapy with docetaxel or anti-PD-L1 showed only partial inhibition of tumor growth, while 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 relative to the reduction in tumor growth obtained with TH1902 alone. Both docetaxel and TH1902 appeared to be well tolerated, as mouse body weight was barely affected when compared to the control group ( Figure 7DHowever, anti-PD-L1 alone or in combination with docetaxel or TH1902 produced similar weight loss, suggesting that the addition of TH1902 had a limited effect on mouse body weight ( Figure 7D ). Taken together, these data support the therapeutic efficiency of TH1902 as a monotherapy, which was significantly improved when combined with anti-PD-L1. The study also showed that TH1902 reversed tumor resistance to CPIs.

[0356] These results were unexpected, as B16-F10 melanomas are generally considered immune-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 ]).

[0357] Example 9: Effect of combining different levels of TH1902 with anti-PD-L1 on tumor growth in a survival study.

[0358] Next, survival studies were performed on mice treated with (or without) anti-PD-L1 alone and with different doses of TH1902. Figure 7CThe results depicted in show that TH1902 exhibits a strong dose-dependent inhibition of tumor growth. Relative to animals treated with the vehicle, treatment with the lowest dose of TH1902 or anti-PD-L1 alone produced a slight but not significant tumor growth inhibition (TGI) (p=0.1394 and 0.1787, respectively); Tumor size was halved 10 days after the start of treatment, which was similar to the tumor size in animals treated with the vehicle, with a median survival of 14 days (TH1902 4.37 mg / kg / wk and anti-PD-L1, two p=0.0521) or 11.5 days (vehicle). However, the tumors in animals treated with higher doses of TH1902 were significantly smaller, with a TGI of 95% (8.75 mg / kg / wk) or a regression of 54% relative to the initial tumor volume (17.5 mg / kg / wk) at the start of treatment, and a median survival of 17 days and 24 days, respectively. Mice in the survival study showed much better tolerance to anti-PD-L1 than those in the efficacy study. No group of mice in this study experienced significant weight loss, but mice that did not receive intraperitoneal injections of control IgG or anti-PD-L1 mAb appeared to gain more weight; this may be due to the rapid exponential growth of some tumors. Survival curves were generated for each of the nine groups of mice. The two groups that received 17.5 mg / kg / wk TH1902 (with or without anti-PD-L1) showed the longest median survival: 24 and 32.5 days, respectively ( Figure 7B ). Statistically, animals treated with higher concentrations of TH1902 (8.75 and 17.5 mg / kg / wk) as monotherapy and all three combined TH1902 doses (4.37, 8.75, and 17.5 mg / kg / wk) and anti-PD-L1 showed survival curves that were significantly different from those of vehicle-treated animals (Table 2). In addition, the survival curves for all three of these combined TH1902 doses were significantly different from those of the group treated with TH1902 alone (p<0.05), with lifespans increasing by 4.5 versus 2.5, 10 versus 5.5, and 21 versus 12.5 days, respectively, relative to the vehicle (Table 2). The [TH1902 17.5 mg / kg / wk / anti-PD-L1] combination significantly and synergistically increased animal survival compared to either anti-PD-L1 or TH1902 17.5 mg / kg / wk as single agents (median survival increased by 21 days compared to 2.5 days for anti-PD-L1 alone and 12.5 days for TH1902 17.5 mg / kg / wk alone).

[0359] Table 2: Results of survival studies in the B16-F10 melanoma xenograft tumor model.

[0360]

[0361]

[0362] 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.

[0363] Cellular senescence is an irreversible cell cycle arrest that represents a major obstacle to tumorigenesis

[34] . Docetaxel has been reported to induce senescence in mouse lung and prostate tumor cells [35,36], in part through a persistent DNA damage response

[37] . Recently, DNA damage states have been shown to activate the cGAS (cyclic GMP-AMP synthase) / STING (stimulator of interferon genes) pathway in TNBC cells, a pathway that is associated with cell proliferation and invasiveness [38,39]. Given the ability of TH1902 to trigger senescence in B16-F10 melanoma cells and to inhibit cell proliferation in melanoma (as shown herein) and TNBC cells [5], the involvement of the STING pathway was next addressed. Cell lysates were harvested after treatment with docetaxel or TH1902; STING protein expression was assessed by immunoblotting ( Figure 9A Although docetaxel induced STING expression in a densitometric manner, TH1902 was indeed more effective at concentrations up to 500 nM ( Figure 9B In the evaluation of downstream effectors of the STING pathway ( Figure 9C ), TH1902 was found to trigger the expression of the p53 transcription factor, which is known to be involved in the cGAS / STING pathway and cellular senescence [40,41]; TH1902 also triggered the expression of p21, which is known to maintain the viability of senescent cells induced by DNA damage

[42] ( Figure 9D , gray bars). Consistent with activation of the STING pathway, a greater increase in the phosphorylation status of TBK1 and p65 was also observed with TH1902 treatment relative to docetaxel ( Figure 9D TBK1 is an important serine / threonine protein kinase that is known to mediate NF-κB signaling and regulate inflammatory cytokine production and activation of innate immunity

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

[44] .

[0364] We next assessed cytokine regulation in cells treated with docetaxel and TH1902. Activation of the STING pathway was further associated with increased transcript levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF) α produced by TH1902 ( Figure 9EFinally, PD-L1 ( Figure 9F , bottom panel) and MHC-I ( Figure 9F , top panel) were induced at the cell surface of TH1902-treated B16-F10 melanoma cells, both of which were induced by gamma interferon ( Figure 9F , right panel). Upregulation of MHC class I, mediated by NF-κB, is believed to enhance T cell activation

[45] ; this may increase tumor cell recognition, ultimately making melanoma cells more susceptible to cytotoxic CD8 + T cell killing. These data indicate that increased NF-κB expression by phosphorylation of p65 is also associated with a good response to CPI therapy in patients with melanoma [46, 47]. Therefore, TH1902 has the potential to increase anti-tumor immunity, and these data underscore in vitro molecular evidence indicating that compensatory upregulation of the CPI ligand PD-L1 may benefit from the counteraction produced by anti-PD-L1 to achieve maximum anti-tumor immunity in vivo, which is consistent with the synergistic effect obtained with the combination of TH1902 and anti-PD-L1 in Example 9.

[0365] Example 11: TNFα, but not IL-6, triggers PD-L1 and MHC-I cell surface expression in B16-F10 melanoma cells.

[0366] To understand the potential crosstalk that may link TNFα / IL-6 induction triggered by TH1902 with 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 h. After TNFα treatment, a dose-dependent increase in the cell surface expression of PD-L1 and MHC-I was observed ( Figure 10A ), while the cell surface expression of these two proteins remained unchanged after IL-6 treatment ( Figure 10B These data suggest that TNFα, rather than IL-6, is the potential major trigger for the induction of PD-L1 and MHC-I cell surface expression mediated by TH1902.

[0367] Example 12: Effect of TH1902 in an immune-cold lung cancer model.

[0368] The effect of TH1902 on tumor growth was evaluated in another immunosuppressive SORT1-expressing, murine lung carcinoma (LL / 2) syngeneic model. 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, Figure 11A and 11B ). Figure 11C The results depicted in show that treatment with TH1902 induced an almost complete inhibition of LL / 2 tumor growth.

[0369] The results presented herein demonstrate that conjugates comprising peptides targeting Sortilin linked to chemotherapeutic agents allow for the additional induction of immune cell infiltration and cytotoxic cell-mediated tumor killing in immunologically cold tumors, and that combining such conjugates with immune checkpoint inhibitors allows for the eradication of tumors, including tumors that are resistant to treatment with immune checkpoint inhibitor monotherapy.

[0370] Although the present invention has been described above by way of specific embodiments thereof, modifications may be made thereto without departing from the spirit and essence of the invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including but not limited to." The singular forms "a," "an," and "the" include the corresponding plural referents unless the context clearly dictates otherwise.

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Claims

1. A method for (i) enhancing an anti-tumor immune response in a subject suffering from a cancer expressing Sortilin and / or (ii) treating a subject suffering from a cancer expressing Sortilin that is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound is of formula A-(B) n ,in A is a peptide compound of 30 residues or less comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13: 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) in 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 independently selected from any amino acid; X 16 、X 17 、X 20 and X 21 independently selected from Q, P, Y, I and L; n is 0, 1, 2, 3, 4, or 5; When X9 is present more than once, each of said X9 is independently selected from any amino acid; When X 19 When present more than once, each of said X9 is independently selected from any amino acid, Optionally, the peptide compound is cyclic, B is at least one therapeutic agent, wherein B is linked 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 to the amino acid sequence of any one of SEQ ID NOs: 1-13. 3 . The method according to claim 1 , wherein the peptide compound comprises the amino acid sequence of any one of SEQ ID NOs: 1-13, and further comprises 1 to 3 additional amino acids at its amino and / or carboxyl termini. The method according to claim 3 , wherein the peptide compound comprises a cysteine ​​residue at its amino terminus and / or carboxyl terminus.

5. The method of claim 4, wherein the peptide compound comprises 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), Where X1-X 21 As defined in claim 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. The method according to claim 1 or 5 , wherein the peptide compound comprises the amino acid sequence of SEQ ID NO: 1 or 2.

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

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

5.

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

6.

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

7.

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

8.

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

9.

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

10.

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 according to claim 1 or 5, wherein the peptide compound comprises the amino acid sequence of SEQ ID NO:

11.

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

12.

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

13.

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

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

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

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

23. The method according to any one of claims 1 to 22, wherein the conjugate is represented by SEQ ID NO: 23 or SEQ ID NO: 24: GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(SEQ ID NO:22); Acetyl-GVRAK(J 1 )AGVRN(Nle)FK(J 2 )SESY(SEQ ID NO:23); Among them J 1 and J 2 Each is independently a therapeutic agent linked 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. The method of claim 24, wherein the chemotherapeutic agent is a taxane.

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

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. 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. 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. 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. Use of a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, for the preparation 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. The use according to claim 34 or 35, wherein the conjugate compound, a pharmaceutically acceptable salt thereof or a drug is used in combination with immunotherapy.

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

38. The use according to 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. 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. A conjugate compound as defined in any one of claims 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.

44. The conjugate compound for use according to claim 43, or a pharmaceutically acceptable salt thereof, wherein the peptide compound is used in combination with immunotherapy.

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

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

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

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

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

51. A method for treating a Sortilin-expressing cancer in a subject, the method 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. The method of claim 51, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

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

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

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

60. 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 treatment of a cancer expressing Sortilin.

61. 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 preparation of a medicament for the treatment of a cancer expressing Sortilin.

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

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

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

70. A combination therapy comprising a conjugate compound as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and immunotherapy, for treating a cancer expressing Sortilin.

71. The combination therapy for use of claim 70, wherein the immunotherapy comprises immune checkpoint inhibitor (ICI) therapy.

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

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

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

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. The 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 in different compositions.

78. The 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 in the same composition.

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