New tumor immune intervention target SIDT1, inhibitor thereof and application of new tumor immune intervention target SIDT1 in tumor resistance

Abstract

According to the invention, a novel immune checkpoint molecule SIDT1 is found. The SIDT1 is a powerful T cell inhibition factor mainly expressed on CD8 < + > T cells. A series of SIDT1 inhibitors are identified in the invention. The SIDT1 inhibitor can enhance the anti-tumor CD8T cell reaction and limit tumor progression, so that a new thought is provided for treatment and prevention of related tumor patients with PD-1 treatment resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy of tumors. Specifically, the present application relates to a new target of tumor immunointervention, SIDT1, and its inhibitors and the application in anti-tumor. BACKGROUND

[0002] Immune checkpoint blockade therapy, particularly anti-PD-1 / PD-L1 therapy, has brought revolutionary changes in the treatment of multiple cancer types. However, only a small fraction of patients with solid tumors or certain hematological malignancies respond to anti-PD-1 therapy. Tumor resistance to immunotherapy remains a major challenge. The main role of anti-PD-1 therapy is to block the PD-1 / PD-L1 T cell inhibitory pathway in the tumor microenvironment (TME), and therefore, the weak or lack of PD-L1 ligand expression on tumor cells or stromal cells in the TME constitutes a direct resistance mechanism. In addition, anti-PD responses are more pronounced in "hot" tumors with significant T cell responses, and T cells are the main cell type expressing the PD-1 checkpoint receptor.

[0003] PD-1 is a checkpoint receptor expressed on a subset of T cells with "exhausted" phenotype, which often co-expresses LAG-3 or TIM-3. The interaction of PD-1 receptor on T cells with PD-L1 of tumor cells or myeloid cells inhibits anti-tumor T cell immunity by impairing T cell activation, proliferation and effector function. PD-1 / PD-L1 blockade is believed to repair or functionally restore these "exhausted" T cells, thereby controlling tumor growth. In this regard, many clinical programs are focusing on co-targeting PD-1 / PD-L1 with other checkpoint receptors on exhausted T cells. However, in addition to the PD-1 / LAG-3 combination, which has been approved by the FDA for melanoma, most of these combinations have not produced meaningful clinical signals. These results collectively indicate the need to identify key pathways beyond the regulation of exhausted T cells to restore anti-tumor T cell immunity.

[0004] Therefore, there is an urgent need in the art to discover new targets of tumor immunointervention in order to restore anti-tumor T cell immunity. SUMMARY

[0005] The purpose of the present application is to provide a new target of tumor immunointervention.

[0006] Another purpose of the present application is to provide inhibitors of the above-mentioned new target of tumor immunointervention, including macromolecular inhibitors and small molecule inhibitors.

[0007] Another purpose of the present application is to provide the application of the above-mentioned new target of tumor immunointervention and its inhibitors in anti-tumor.

[0008] In a first aspect, the present application provides the use of SIDT1 as an immune checkpoint.

[0009] In preferred embodiments, the SIDT1 is human SIDT1.

[0010] In preferred embodiments, the amino acid sequence of the human SIDT1 (Uniprot: Q9NXL6-2) is as set forth in SEQ ID NO: 1 below:

[0011] MRGCLRLALLCALPWLLLAASPGHPAKSPRQPPAPRRDPFDAARGADFDHVYSGVVNLSTENIYSFNYTSQPDQVTAVRVYVNSSSENLNYPVLVVVRQQKEVLSWQVPLLFQGLYQRSYNYQEVSRTLCPSEATNETGPLQQLIFVDVASMAPLGAQYKLLVTKLKHFQLRTNVAFHFTASPSQPQYFLYKFPKDVDSVIIKVVSEMAYPCSVVSVQNIMCPVYDLDHNVEFNGVYQSMTKKAAITLQKKDFPGEQFFVVFVIKPEDYACGGSFFIQEKENQTWNLQRKKNLEVTIVPSIKESVYVKSSLFSVFIFLSFYLGCLLVGFVHYLRFQRKSIDGSFGSNDGSGNMVASHPIAASTPEGSNYGTIDESSSSPGRQMSSSDGGPPGQSDTDSSVEESDFDTMPDIESDKNIIRTKMFLYLSDLSRKDRRIVSKKYKIYFWNIITIAVFYALPVIQLVITYQTVVNVTGNQDICYYNFLCAHPLGVLSAFNNILSNLGHVLLGFLFLLIVLRRDILHRRALEAKDIFAVEYGIPKHFGLFYAMGIALMMEGVLSACYHVCPNYSNFQFDTSFMYMIAGLCMLKLYQTRHPDINASAYSAYASFAVVIMVTVLGVVFGKNDVWFWVIFSAIHVLASLALSTQIYYMGRFKIDVSDTDLGIFRRAAMVFYTDCIQQCSRPLYMDRMVLLVVGNLVNWSFALFGLIYRPRDFASYMLGIFICNLLLYLAFYIIMKLRSSEKVLPVPLFCIVATAVMWAAALYFFFQNLSSWEGTPAESREKNRECILLDFFDDHDIWHFLSATALFFSFLVLLTLDDDLDVVRRDQIPVF.

[0012] In a specific embodiment, the use includes, but is not limited to, development or preparation of a medicament for treating or preventing a SIDT1 -related disease, development or preparation of a diagnostic reagent for a SIDT1 -related disease.

[0013] In a preferred embodiment, the medicament is a SIDT1 -inhibiting or SIDT1 -knocking out immune effector cell.

[0014] In preferred embodiments, the immune effector cell is a genetically engineered immune effector cell; including but not limited to CAR T cells, CAR NK cells, TCR T cells.

[0015] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: T cells, NK cells, plasma cells, APSC pluripotent cells, mast cells, macrophages, TIL cells.

[0016] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: CD8+ T cells, CD4+ T cells, Treg cells, NKT cells, TIL cells.

[0017] In preferred embodiments, the CD8+ T cell is a PD-1 negative CD8+ T cell.

[0018] In specific embodiments, the SIDT1 -related disease is a tumor with elevated SIDT1 expression; preferably, the tumor includes but is not limited to colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; more preferably, the tumor is colorectal cancer or prostate cancer.

[0019] In a second aspect, the present application provides use of SIDT1 as a prognostic marker for a SIDT1 -related disease.

[0020] In preferred embodiments, the SIDT1 is SIDT1 expressed in CD8+ T cells, in particular PD-1 negative CD8+ T cells.

[0021] In preferred embodiments, the SIDT1 -related disease is a tumor with elevated SIDT1 expression.

[0022] In preferred embodiments, the tumor includes but is not limited to colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0023] In preferred embodiments, the prognostic marker is a marker for tumor survival period.

[0024] In a third aspect, the present application provides use of a SIDT1 inhibitor in the manufacture of a medicament for preventing or treating a SIDT1 -related disease, or a diagnostic reagent for a SIDT1 -related disease.

[0025] In preferred embodiments, the SIDT1 -related disease is a tumor with elevated SIDT1 expression.

[0026] In preferred embodiments, the tumor comprises, but is not limited to, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0027] In preferred embodiments, the SIDT1 inhibitor is a substance that inhibits the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0028] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0029] In preferred embodiments, the SIDT1 inhibitor comprises a macromolecular inhibitor or a small molecule inhibitor.

[0030] In preferred embodiments, the macromolecular inhibitor comprises an antibody, an antisense nucleic acid, a polypeptide inhibitor or an ADC.

[0031] In preferred embodiments, the antisense nucleic acid comprises a miRNA or an siRNA.

[0032] In preferred embodiments, the small molecule inhibitor is a compound that is capable of binding to the E555, S559 and D574 residues of SIDT1.

[0033] In preferred embodiments, the small molecule inhibitor is a compound selected from the group consisting of:

[0034] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0035] Preferably, the small molecule inhibitor is Relamorelin (TFA).

[0036] In a fourth aspect, the present application provides an inhibitor of SIDT1.

[0037] In preferred embodiments, the SIDT1 inhibitor is a substance that inhibits the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0038] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0039] In preferred embodiments, the SIDT1 inhibitor comprises a macromolecular inhibitor or a small molecule inhibitor.

[0040] In preferred embodiments, the macromolecular inhibitor comprises an antibody, an antisense nucleic acid, a polypeptide inhibitor or an ADC.

[0041] In preferred embodiments, the antisense nucleic acid comprises a miRNA or an siRNA.

[0042] In preferred embodiments, the small molecule inhibitor is a compound that is capable of binding to the E555, S559 and D574 residues of SIDT1.

[0043] In preferred embodiments, the small molecule inhibitor is a compound selected from the group consisting of:

[0044] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0045] Preferably, the small molecule inhibitor is Relamorelin (TFA).

[0046] In a fifth aspect, the present application provides a pharmaceutical composition comprising:

[0047] 1) a SIDT1 inhibitor; or

[0048] 2) a SIDT1 knockout immune effector cell; or

[0049] 3) a SIDT1 inhibitor and a SIDT1 non-knockout immune effector cell; or

[0050] 4) a SIDT1 inhibitor and a SIDT1 knockout immune effector cell.

[0051] In preferred embodiments, the pharmaceutical composition further comprises one or more other immune checkpoint inhibitors.

[0052] In preferred embodiments, the other immune checkpoint inhibitor comprises, but is not limited to, an inhibitor of PD-1 / PD-L1, CTLA4, LAG3, CD4, CD40, CD80, TNF, CD86, 4-1BB / CD137, B7-H3 / CD276, LMTK3, TIM-3, IDO1; preferably a PD-1 or PD-L1 inhibitor.

[0053] In preferred embodiments, the immune effector cell is a genetically engineered immune effector cell.

[0054] In preferred embodiments, the genetically engineered immune effector cell is a CAR T cell, a CAR NK cell, a TCR T cell.

[0055] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: a T cell, a NK cell, a plasmacytic cell, an APSC pluripotent cell, a mast cell, a macrophage, a TIL cell.

[0056] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: a CD8+ T cell, a CD4+ T cell, a Treg cell, a NKT cell, a TIL cell.

[0057] In preferred embodiments, the immune effector cell is a PD-1 negative CD8+ T cell.

[0058] In preferred embodiments, the SIDT1 inhibitor is a substance that inhibits the Ca 2+ In combination with the function of binding and / or the function of Ca 2+ transporting.

[0059] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ In combination with the function of binding and / or the function of Ca 2+ transporting.

[0060] In preferred embodiments, the SIDT1 inhibitor comprises a macromolecular inhibitor or a small molecule inhibitor.

[0061] In preferred embodiments, the macromolecular inhibitor comprises an antibody, an antisense nucleic acid, a polypeptide inhibitor, or an ADC.

[0062] In preferred embodiments, the antisense nucleic acid comprises a miRNA or an siRNA.

[0063] In preferred embodiments, the small molecule inhibitor is a compound capable of binding to the E555, S559, and D574 residues of SIDT1.

[0064] In preferred embodiments, the small molecule inhibitor is the following compound or a pharmaceutically acceptable salt thereof:

[0065] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0066] Preferably, the small molecule inhibitor is Relamorelin (TFA).

[0067] In a sixth aspect, the present application provides a combination of a SIDT1 inhibitor with other immune checkpoint inhibitors.

[0068] In preferred embodiments, the other immune checkpoint inhibitors include, but are not limited to, inhibitors of PD-1 / PD-L1, CTLA4, LAG3, CD4, CD40, CD80, TNF, CD86, 4-1BB / CD137, B7-H3 / CD276, LMTK3, TIM-3, IDO1; preferably a PD-1 or PD-L1 inhibitor.

[0069] In preferred embodiments, the combination further comprises one or more immune effector cells or SIDT1 knockout immune effector cells.

[0070] In preferred embodiments, the immune effector cell is a genetically engineered immune effector cell.

[0071] In preferred embodiments, the genetically engineered immune effector cell is a CAR T cell, a CAR NK cell, a TCR T cell.

[0072] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: T cells, NK cells, plasma cells, APSC pluripotent cells, mast cells, macrophages, TIL cells.

[0073] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: CD8+ T cells, CD4+ T cells, Treg cells, NKT cells, TIL cells.

[0074] In preferred embodiments, the immune effector cell is a PD-1 negative CD8+ T cell.

[0075] In preferred embodiments, the SIDT1 inhibitor is a substance that inhibits the Ca 2+ binds to and / or inhibits the transport function of SIDT1. 2+ binds to and / or inhibits the transport function of SIDT1.

[0076] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ binds to and / or inhibits the transport function of SIDT1. 2+ binds to and / or inhibits the transport function of SIDT1.

[0077] In preferred embodiments, the SIDT1 inhibitor comprises a macromolecular inhibitor or a small molecule inhibitor.

[0078] In preferred embodiments, the macromolecular inhibitor comprises an antibody, an antisense nucleic acid, a polypeptide inhibitor or an ADC.

[0079] In preferred embodiments, the antisense nucleic acid comprises a miRNA or an siRNA.

[0080] In preferred embodiments, the small molecule inhibitor is a compound that is capable of binding to the E555, S559 and D574 residues of SIDT1.

[0081] In preferred embodiments, the small molecule inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0082] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0083] Preferably, the SIDT1 inhibitor is Relamorelin (TFA).

[0084] In a seventh aspect, the present application provides use of the SIDT1 inhibitor of the sixth aspect in combination with other immune checkpoint inhibitors in the manufacture of a medicament for a SIDT1 related disease.

[0085] In a preferred embodiment, the SIDT1 related disease is a tumor with increased SIDT1 expression.

[0086] In a preferred embodiment, the tumor comprises, but is not limited to, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0087] In an eighth aspect, the present application provides a method of enhancing or restoring immune effector cell function, the method comprising the step of contacting a SIDT1 inhibitor with an immune effector cell; or,

[0088] the step of knocking out SIDT1 of the immune effector cell.

[0089] In a preferred embodiment, the immune effector cell is one or more selected from the group consisting of T cells, NK cells, plasma cells, APSC pluripotent cells, mast cells, macrophages, TIL cells.

[0090] In a preferred embodiment, the immune effector cell is one or more selected from the group consisting of CD8+ T cells, CD4+ T cells, Treg cells, NKT cells, TIL cells.

[0091] In a preferred embodiment, the immune effector cell is a PD-1 negative CD8+ T cell.

[0092] In a preferred embodiment, the immune effector cell is a genetically engineered immune effector cell.

[0093] In preferred embodiments, the genetically engineered immune effector cell is a CAR T cell, a CAR NK cell, a TCR T cell.

[0094] In preferred embodiments, the enhancing immune effector cell function includes, but is not limited to, enhancing the anti-tumor immune response of the immune effector cell; promoting the activation and proliferation of the immune effector cell, particularly in the tumor microenvironment (TME).

[0095] In preferred embodiments, the method is an in vitro method.

[0096] In preferred embodiments, the SIDT1 inhibitor is a macromolecular inhibitor or a small molecule inhibitor. 2+ In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the Ca 2+ transporting function of SIDT1.

[0097] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ transporting function of SIDT1. 2+ transporting function of SIDT1.

[0098] In preferred embodiments, the SIDT1 inhibitor includes a macromolecular inhibitor or a small molecule inhibitor.

[0099] In preferred embodiments, the macromolecular inhibitor includes an antibody, an antisense nucleic acid, a polypeptide inhibitor, or an ADC.

[0100] In preferred embodiments, the antisense nucleic acid includes a miRNA or an siRNA.

[0101] In preferred embodiments, the small molecule inhibitor is a compound that is capable of binding to the E555, S559 and D574 residues of SIDT1.

[0102] In preferred embodiments, the small molecule inhibitor is a compound or a pharmaceutically acceptable salt thereof:

[0103] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0104] Preferably, the Relamorelin (TFA).

[0105] In preferred embodiments, the method further comprises the step of contacting the immune effector cell with one or more additional immune checkpoint inhibitors.

[0106] In preferred embodiments, the additional immune checkpoint inhibitor comprises, but is not limited to, an inhibitor of PD-1 / PD-L1, CTLA4, LAG3, CD4, CD40, CD80, TNF, CD86, 4-1BB / CD137, B7-H3 / CD276, LMTK3, TIM-3, IDO1; preferably a PD-1 or PD-L1 inhibitor.

[0107] In a ninth aspect, the present application provides the use of a SIDT1 knockout immune effector cell in the manufacture of a medicament for the treatment of a tumor.

[0108] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: a T cell, a NK cell, a plasmacytic cell, an APSC pluripotent cell, a mast cell, a macrophage, a TIL cell.

[0109] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: a CD8+ T cell, a CD4+ T cell, a Treg cell, a NKT cell, a TIL cell.

[0110] In preferred embodiments, the immune effector cell is a PD-1 negative CD8+ T cell.

[0111] In preferred embodiments, the immune effector cell is a genetically engineered immune effector cell.

[0112] In preferred embodiments, the genetically engineered immune effector cell is a CAR T cell, a CAR NK cell, a TCR T cell.

[0113] In a tenth aspect, the present application provides a method of treating a SIDT1 -related disease, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a SIDT1 inhibitor; or,

[0114] the step of administering to a subject in need thereof a therapeutically effective amount of one or more SIDT1 -knockout immune effector cells; or,

[0115] the step of administering to a subject in need thereof a therapeutically effective amount of a SIDT1 inhibitor and a therapeutically effective amount of one or more non-SIDT1 -knockout immune effector cells.

[0116] In preferred embodiments, the SIDT1 -related disease is a tumor with elevated SIDT1 expression.

[0117] The tumor includes, but is not limited to, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0118] In preferred embodiments, the SIDT1 inhibitor is a substance that inhibits the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0119] In preferred embodiments, the SIDT1 inhibitor is a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ binding function and / or the Ca 2+ transport function of SIDT1.

[0120] In preferred embodiments, the SIDT1 inhibitor comprises a macromolecular inhibitor or a small molecule inhibitor.

[0121] In preferred embodiments, the macromolecular inhibitor comprises an antibody, an antisense nucleic acid, a polypeptide inhibitor or an ADC.

[0122] In preferred embodiments, the antisense nucleic acid comprises a miRNA or an siRNA.

[0123] In preferred embodiments, the small molecule inhibitor is a compound capable of binding to the E555, S559 and D574 residues of SIDT1.

[0124] In preferred embodiments, the small molecule inhibitor is a compound of the following formula:

[0125] Iodixanol, Acarbose, Peptide T, Proanthocyanidins, Batefenterol, Difelikefalin, Ibutamoren (Mesylate), Diosmin, Capreomycin (sulfate), Glesatinib (hydrochloride), Vilanterol, Vilanterol (trifenatate), Relamorelin (TFA), Lapatinib ditosylate, Lapatinib, Hesperidin;

[0126] Preferably, the Relamorelin (TFA).

[0127] In preferred embodiments, the method further comprises the step of administering a therapeutically effective amount of one or more additional immune checkpoint inhibitors.

[0128] In preferred embodiments, the additional immune checkpoint inhibitors include, but are not limited to, inhibitors of PD-1 / PD-L1, CTLA4, LAG3, CD4, CD40, CD80, TNF, CD86, 4-1BB / CD137, B7-H3 / CD276, LMTK3, TIM-3, IDO1; preferably a PD-1 or PD-L1 inhibitor.

[0129] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: T cells, NK cells, plasma cells, APSC pluripotent cells, mast cells, macrophages, TIL cells.

[0130] In preferred embodiments, the immune effector cell is one or more selected from the group consisting of: CD8+ T cells, CD4+ T cells, Treg cells, NKT cells, TIL cells.

[0131] In preferred embodiments, the immune effector cell is a PD-1 negative CD8+ T cell.

[0132] In preferred embodiments, the immune effector cell is a genetically engineered immune effector cell.

[0133] In preferred embodiments, the genetically engineered immune effector cell is a CAR T cell, a CAR NK cell, a TCR T cell.

[0134] In an eleventh aspect, the present application provides a method for prognosis of a SIDT1 -related disease, the method comprising the steps of:

[0135] 1) detecting the expression level of SIDT1 in CD8+ T cells from the subject; and

[0136] 2) assessing the prognosis of the subject according to the expression level of SIDT1 detected in step 1).

[0137] In a preferred embodiment, the SIDT1 -related disease is a tumor with increased expression of SIDT1.

[0138] In a preferred embodiment, the tumor includes, but is not limited to, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0139] In a preferred embodiment, the assessing the prognosis of the subject is assessing the survival of the tumor.

[0140] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0141] Figure 1 shows the discovery process of the novel immune checkpoint SIDT1 expressed in PD-1 negative CD8+ T cell population;

[0142] Figure 2 shows that Sidt1 negatively regulates CD8+ T cell-mediated anti-tumor immune response;

[0143] Figure 3 shows that SIDT1 regulates the function of cytotoxic T lymphocytes (CTLs) by modulating the Ca 2+ level in the cytoplasm;

[0144] Figure 4 shows that SIDT1 is a cytotoxic T lymphocyte-specific Ca 2+ transporter;

[0145] Figure 5 shows that Relamorelin (TFA) enhances CD8+ T cell function to inhibit tumor growth by targeting SIDT1. DETAILED DESCRIPTION

[0146] To solve the technical problems existing in the art, the present inventors have unexpectedly found a new immune checkpoint molecule SIDT1. The present inventors have also found a series of small molecule inhibitors, such as Relamorelin (TFA), for use in the preparation of a medicament or preparation for preventing and / or treating SIDT1-related tumor immunity. The SIDT1-related tumor immunity refers to a related disease in which CD8+ T cell function is impaired due to high expression of SIDT1, including colorectal cancer, prostate cancer, and liver cancer. The small molecule inhibitor Relamorelin (TFA) provides a new method for the prevention and treatment of SIDT1-related diseases. The specific mechanism of the small molecule inhibitor Relamorelin (TFA) in treating SIDT1-related diseases is that Relamorelin (TFA) directly binds to SIDT1 and inhibits its transport Ca 2+ , regulates the activation of CD8+ T cells and the effect function achieved. Relamorelin (TFA) can enhance the killing function of CD8+ T cells in vivo and in vitro, and CD8+ T cells treated with Relamorelin (TFA) have better efficacy in tumor-bearing mice. In addition, CAR-T treated with Relamorelin (TFA) has stronger killing effect and can enhance the efficacy of CAR-T. Therefore, Relamorelin (TFA) provides a new idea for the prevention and treatment of SIDT1-related tumor immunity. On this basis, the present application is completed.

[0147] immune effector cell

[0148] The term "immune effector cell" used herein has the same meaning as understood by those skilled in the art, and refers to an immune cell that participates in the clearance of foreign antigens and exerts an effector function in an immune response.

[0149] For example, in T cells, CD8+ T cells play a crucial role in tumor immunity because they are a population of cytotoxic T cells that can detect and eliminate tumor cells. Although PD-1 high expression CD8 T cells represent exhausted T cells, many tumor-infiltrating T cells are negative for PD-1. The function and clinical significance of these PD-1 low expression and PD-1 high expression CD8+ T cells have not been well characterized. In a recent report, a high proportion of PD-1 low expression CD8+ T cells was associated with improved disease-free survival. This suggests that rescuing PD-1 low expression cells can be a mechanism for clinical benefit and can serve as a more accurate clinical response biomarker for anti-PD-1 targeted immunotherapy.

[0150] In a specific embodiment, the immune effector cell described in the present application is a T cell, a NK cell, a plasma cell, an APSC pluripotent cell, a mast cell, a macrophage or a TIL cell. In a preferred embodiment, the immune effector cell described in the present application is a CD8+ T cell, a CD4+ T cell, a Treg cell, a NKT cell or a TIL cell. More preferably, the immune effector cell described in the present application is a CD8+ T cell that is PD-1 negative.

[0151] The immune effector cell described in the present application can also be a genetically engineered immune effector cell, such as a CAR T cell, a CAR NK cell, a TCR T cell.

[0152] Immune checkpoint and its inhibitors of the present application

[0153] The inventors of the present application have characterized the lineage of PD-1 negative and PD-1 positive CD8+ T cells in tumor infiltrating T cells from single cell sequencing data. The inventors of the present application have identified and characterized a novel immune checkpoint molecule, SIDT1, which is highly and specifically expressed on CD8+ T cells. In addition, SIDT1 is considered a risk gene for prostate cancer. SIDT1 is a multi-pass transmembrane protein with high homology, similar to SID-1 (Systemic Interference Deficient protein 1) of Caenorhabditis elegans. SIDT1 is localized to the plasma membrane and is believed to be involved in RNA transport, cholesterol transport, hydrolytic enzyme activity, and the function of glycosylated RNA, but its specific function in T cells has not been proposed.

[0154] The present application discloses SIDT1 as a potential calcium transporter, which regulates cytosolic Ca 2+ homeostasis in CD8+ T cells. Knockout of SIDT1 enhances the anti-tumor immune response of CD8+ T cells. Therefore, we screened for small molecule inhibitors of this new target of tumor immunity and obtained an effective small molecule inhibitor Relamorelin (TFA), which was found to be useful for tumor immunotherapy.

[0155] Based on the teachings of the present application, it will be appreciated by those skilled in the art that the SIDT1 inhibitors described herein are substances that inhibit the Ca 2+ binding function and / or Ca 2+ transport function of SIDT1. For example, the SIDT1 inhibitor can be a substance that binds to the E555, S559 and D574 residues of SIDT1, thereby inhibiting the Ca 2+ binding function and / or Ca 2+ transport function of SIDT1.

[0156] As can be reasonably contemplated by one skilled in the art, the SIDT1 inhibitor can include a macromolecular inhibitor or a small molecule inhibitor. For example, the macromolecular inhibitor includes an antibody, an antisense nucleic acid, a polypeptide inhibitor, or an ADC. In a specific embodiment, the antisense nucleic acid includes a miRNA or an siRNA. The small molecule inhibitor is a compound capable of binding to the E555, S559, and D574 residues of SIDT1. In a specific embodiment, the small molecule inhibitor is a compound shown in the following table or a pharmaceutically acceptable salt thereof:

[0157]

[0158]

[0159]

[0160]

[0161] Relamorelin (TFA) is preferred.

[0162] Pharmaceutical composition of the present application

[0163] Based on the SIDT1 inhibitor of the present application, the present application also provides a pharmaceutical composition for treating a SIDT1 -related disease, such as a tumor with increased expression of SIDT1. In a specific embodiment, the tumor includes, but is not limited to, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0164] Based on the teachings of the present application, one skilled in the art can understand that the pharmaceutical composition of the present application can comprise a SIDT1 inhibitor and a pharmaceutically acceptable excipient. One skilled in the art can also reasonably understand that one or more other immune checkpoint inhibitors can also be included in the pharmaceutical composition of the present application. For example, the other immune checkpoint inhibitors include, but are not limited to, inhibitors of PD-1 / PD-L1, CTLA4, LAG3, CD4, CD40, CD80, TNF, CD86, 4-1BB / CD137, B7-H3 / CD276, LMTK3, TIM-3, IDO1; preferably, a PD-1 or PD-L1 inhibitor.

[0165] Based on the teachings of the present application, one skilled in the art can also understand that knocking out SIDT1 of immune effector cells, particularly CD8+T cells (e.g., PD-1 negative CD8+T cells), can enhance the anti-tumor activity of these immune effector cells. Therefore, the pharmaceutical composition of the present application can comprise immune effector cells with SIDT1 knockout and a pharmaceutically acceptable excipient.

[0166] The SIDT1 inhibitor of the present application can improve or restore the function of immune effector cells. Therefore, the pharmaceutical composition of the present application can also comprise the SIDT1 inhibitor of the present application and one or more non-SIDT1 knockout immune effector cells. The "non-SIDT1 knockout immune effector cell" described herein refers to an immune effector cell that is not SIDT1 knockout, and thus needs to be combined with the SIDT1 inhibitor of the present application to enhance or restore the function. In specific embodiments, the non-SIDT1 knockout immune effector cell includes but is not limited to T cells, NK cells, plasma cells, APSC pluripotent cells, mast cells, macrophages, TIL cells. In particular, the immune effector cell that can be combined with the SIDT1 inhibitor of the present application can be a genetically engineered immune effector cell, so as to enhance the therapeutic effect of the immune cell. For example, the genetically engineered immune effector cell includes but is not limited to CAR T cells, CAR NK cells, TCR T cells.

[0167] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the SIDT1 inhibitor of the present application, other immune checkpoint inhibitors, immune effector cells or genetically engineered immune effector cells, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, with normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical composition such as needle and solution should be manufactured under sterile conditions.

[0168] The amount of active ingredient to be administered is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dose should also take into account the administration route, the patient's health status, etc., which are within the skill of the skilled physician.

[0169] Applications

[0170] As described above, the present inventors found that SIDT1 can act as an immune checkpoint, and thus the present application has wide biological application value and clinical application value, and its application involves multiple fields such as diagnosis and treatment of diseases related to cell therapy, basic medical research, biological research, etc.

[0171] In specific embodiments, the use includes, but is not limited to, the development or preparation of a medicament for treating or preventing a SIDT1 -related disease, the development or preparation of a diagnostic reagent for a SIDT1 -related disease. In preferred embodiments, the SIDT1 -related disease is a tumor with high expression of SIDT1, including but not limited to colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; preferably, the tumor is colorectal cancer or prostate cancer.

[0172] Based on the teachings of the present application, one skilled in the art can understand that the expression level of SIDT1 in immune effector cells indicates whether the immune effector cells can normally exert their functions, and at the same time, indicates the survival ability or survival period of tumor cells. Therefore, the expression level of SIDT1, especially SIDT1 in CD8+ T cells, can be used as a prognostic marker, especially a tumor survival period marker.

[0173] Methods of use

[0174] Based on the findings of the present application that SIDT1 can be used as an immune checkpoint or a prognostic marker, one skilled in the art can conceive methods of enhancing or restoring the function of immune effector cells, methods of treating SIDT1 -related diseases, such as tumors, and methods of prognosis.

[0175] In specific embodiments, the method of enhancing or restoring the function of immune effector cells of the present application comprises the step of contacting a SIDT1 inhibitor with the immune effector cells; or, comprises the step of knocking out SIDT1 of the immune effector cells.

[0176] In specific embodiments, the method of treating a SIDT1 -related disease of the present application comprises the step of administering a therapeutically effective amount of a SIDT1 inhibitor to a subject in need thereof; or,

[0177] the step of administering a therapeutically effective amount of one or more SIDT1 -knocked out immune effector cells to a subject in need thereof; or,

[0178] the step of administering a therapeutically effective amount of a SIDT1 inhibitor and a therapeutically effective amount of one or more non-SIDT1 -knocked out immune effector cells to a subject in need thereof.

[0179] In specific embodiments, the method of prognosis of a SIDT1 -related disease of the present application comprises the following steps:

[0180] 1) detecting the expression level of SIDT1 in CD8+ T cells from the subject; and

[0181] 2) evaluating the prognosis of the subject according to the expression level of SIDT1 detected in step 1).

[0182] Advantages or benefits of the present application:

[0183] 1. Discovery of a new immune checkpoint molecule: The present invention discovers a molecule, SIDT1, which is specifically expressed on CD8+ T cells and is involved in tumor immune regulation, thus discovering a new immune checkpoint molecule;

[0184] 2. Gene knockout enhances anti-tumor immune response: The present invention discovers that by knocking out SIDT1, the anti-tumor immune response of CD8+ T cells can be enhanced;

[0185] 3. The present invention discovers that Relamorelin (TFA) as a small molecule inhibitor of the new immune checkpoint molecule SIDT1 has similar therapeutic effects as knocking out SIDT1, and as a small molecule inhibitor, Relamorelin (TFA) has shown certain potential and advantages in the field of tumor immunotherapy;

[0186] 4. Regulation of cytoplasmic Ca 2+ Homeostasis: The research results of the present invention show that Relamorelin (TFA) can inhibit SIDT1 and regulate cytoplasmic Ca 2+ homeostasis in CD8+ T cells, which helps to enhance the anti-tumor immune response of CD8+ T cells;

[0187] 5. Targeting tumor microenvironment (TME): The present invention discovers that Relamorelin (TFA) may improve the tumor microenvironment, promote the activation and proliferation of T cells, and thus improve the effect of anti-tumor T cell immunity;

[0188] 6. Possible treatment of multiple cancer types: The present invention discovers that although the research results of Relamorelin (TFA) mainly focus on colorectal cancer and prostate cancer tumor immunotherapy, its potential mechanism may be applicable to multiple cancer types, thus providing a new direction for cancer treatment;

[0189] 7. Safety and tolerability: In clinical trials, Relamorelin (TFA) has shown acceptable safety and tolerability, and no adverse events in the heart or neurology have been found, which provides favorable safety support for its use as a tumor immunotherapy drug;

[0190] 8. Possible improvement of T cell response: Since Relamorelin (TFA) can target specific immune checkpoint molecules on T cells, it helps to improve the response of T cells to tumors, especially for those patients who do not respond well to anti-PD-1 therapy;

[0191] 9. Potential clinical applications: The discovery and research progress of Relamorelin (TFA) indicate that it may become a new target for tumor immunotherapy and has potential clinical application value.

[0192] In summary, the discovery of the new immune checkpoint molecule SIDT1 and its small molecule inhibitor Relamorelin (TFA) has laid a new foundation for tumor immunotherapy. Relamorelin (TFA) has shown many advantages in the field of tumor immunotherapy, including targeting a new immune checkpoint molecule, regulating T cell function, improving the tumor microenvironment, and good safety and tolerability. These advantages make it a promising candidate for tumor immunotherapy.

[0193] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the methods are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0194] Example 1. Discovery of a new immune checkpoint SIDT1 expressed in PD-1 negative CD8+ T cell population

[0195] 1. Construction of SIDT1 gene knockout mice

[0196] The Sidt1 gene site was targeted by two sgRNAs targeting the 3rd and 4th exons using CRISPR-Cas9 technology. The primers used for genotyping of Sidt1 knockout mice are as follows:

[0197] Sidt1-5-mut-F: 5'-CTTGGGCTGCTTGTGTTTCG-3' (SEQ ID NO: 2);

[0198] Sidt1-3-mut-F: 5'-GGGGCGTAGTGTGAGATAGC-3' (SEQ ID NO: 3);

[0199] Sidt1-3-mut-R: 5'-AGGGCTCGAACGACCTCACT-3' (SEQ ID NO: 4);

[0200] Sidt1 - / - Pdcd1- / - Sidt1 knockout mice were crossed with OT1 mice to obtain Sidt1 - / - OT1 mice.

[0201] 2. Subcutaneously inoculate tumor-bearing models

[0202] 8-10 week-old male Sidt1 knockout mice, Pdcd1 knockout Sidt1 knockout double knockout mice (Sidt1 - / - Pdcd1 - / - ), were injected with MC38 cells (5x10 5 cells per mouse) or RM-1 cells (1x10 5 cells per mouse) by subcutaneous injection on day 0, respectively. Tumor growth was monitored every two days using an electronic caliper, and was expressed by the average tumor diameter: volume V = π / 6 x length x width 2 On day 16 after treatment, the mice were euthanized, the tumor tissues were isolated, weighed, photographed, and subjected to FACS analysis.

[0203] 3. Experimental results

[0204] The results of Example 1 are shown in Figure 1, in particular, Figure 1 shows the discovery process of the novel immune checkpoint SIDT1 expressed in the PD-1 negative CD8+ T cell population. Among them, Figure 1a shows that reanalysis of single-cell RNA sequencing data from PD-1- and PD-1+ CD8+ T cells of different human cancer patients found that tumor-infiltrating PD-1- CD8+ T cells have different transcriptional signature genes from PD-1+ CD8+ T cells. Figures 1b-1c shows that SIDT1 is the most obvious negative correlation candidate with GZMB / CD8A mRNA levels in various human cancer types. Figure 1d Single-cell RNA sequencing analysis shows that SIDT1 is specifically expressed in CD8+ T cells in the tumor environment. Figure 1e shows the correlation between CTL levels in tumors with different SIDT1 levels and the overall survival rate of patients. Figures 1f-1i shows that SIDT1 knockout mice show significantly slower tumor growth and improved survival rate after inoculation of MC38 tumors compared to normal mice. Figures 1j-1m shows that SIDT1 knockout mice show significantly slower tumor growth and improved survival rate after inoculation of RM-1 tumors compared to normal mice. Figures 1n-1p shows that Sidt1 - / - Pdcd1 - / - mice in the MC38 tumor model compared to Pdcd1 - / -The phenomenon that the tumor growth of mice was significantly slowed down, which indicated that the blockade of SIDT1 significantly improved the effect of anti-PD-1 immunotherapy.

[0205] Therefore, the results of Example 1 showed that SIDT1 specifically expressed on PD-1-CD8+T cells is a new type of immune checkpoint. SIDT1 knockout mice were significantly inhibited in tumor growth in models of colorectal cancer and prostate cancer, and had longer survival time. In addition, SIDT1 can synergize with anti-PD-1 therapy.

[0206] Example 2. Sidt1 negatively regulates CD8+T cell-mediated anti-tumor immune response

[0207] 1. Subcutaneous inoculation of tumor-bearing models

[0208] 8-10 week old male Rag1 gene knockout Sidt1 gene knockout double knockout mice were injected with MC38 cells (5x10 5 cells per mouse) or RM-1 cells (1x10 5 cells per mouse) by subcutaneous injection on day 0. Tumor growth was monitored every two days using an electronic caliper, and the average tumor diameter was used to represent the volume V = π / 6x lengthx width 2 . On day 16 after treatment, the mice were euthanized, the tumor tissue was isolated, weighed, photographed, and subjected to flow analysis.

[0209] 2. Preparation of cytotoxic T lymphocytes (CTLs)

[0210] OT1 Rag1 - / - The spleen cells of the mice were collected and homogenized by sterile treatment. Red blood cells were lysed with red blood cell lysis buffer at room temperature for 5 minutes. The spleen cells were centrifuged and resuspended in T cell culture medium at a concentration of 1x10 6 cells per milliliter (RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin, β-mercaptoethanol), and 10 nM / L (9.6313 ng / mL) OVA 257-264 peptide (Rockland) and 10 ng / mL human recombinant interleukin-2 (Proteintech) were added and cultured for 2 days. Then, the cells were cultured in fresh medium containing 10 ng / mL IL-2 for 2 days for subsequent experiments.

[0211] 3. Adoptive transfer tumor model

[0212] B16-OVA cells (1x10 5 ) were injected subcutaneously into 6-8 week old Rag1 - / -or C57BL / 6J mice. On day 12, tumor-bearing mice with similar tumor sizes were randomly divided into specific groups and were given PBS, wild-type OT1 CTLs, Sidt1 - / - OT1 CTLs (1 x 10 6 ) intravenously, respectively. Tumor growth was monitored every two days using electronic calipers and was expressed as the average tumor diameter: V = π / 6 x length x width 2 .

[0213] 4. Experimental results

[0214] The results of Example 2 are shown in Figure 2. Specifically, Figure 2 shows that Sidt1 negatively regulates CD8+ T cell-mediated anti-tumor immune responses. Therein, Figures 2a-2c shows that Sidt1 - / - Rag1 - / - knockout mice showed no difference after inoculation of MC38 tumors, indicating that immune cells were responsible. Figures 2d-2k shows that intravenous transfer of Sidt1 - / - CTLs to Rag1 + / + or Sidt1 - / - CTLs to C57BL / 6J tumor-bearing B16-OVA mice found that Sidt1 - / - knockout CTLs significantly inhibited tumor growth. Figures 2l-2n shows that Sidt1 - / - Rag1 - / - knockout mice showed no difference after inoculation of RM-1 tumors.

[0215] Thus, the results of Example 2 show that the effect of knocking out SIDT1 on colorectal cancer and prostate cancer is dependent on CD8+ T cells. Subsequent experiments with adoptive transfer of CTLs further confirm this conclusion.

[0216] Example 3. SIDT1 regulates the function of cytotoxic T lymphocytes (CTLs) by modulating Ca 2+ levels in the cytoplasm

[0217] 1. Isolation of tumor infiltrating lymphocytes

[0218] At the end of the experiment, mice were euthanized and tumor tissues were collected for identification of cell-mediated anti-tumor responses. Tumor tissues were minced using surgical scissors and digested in RPMI 1640 medium containing 1 mg / mL Collagenase IV (Sigma-Aldrich, C5138) and 20 pg / mL DNase I (Roche, 11284932001) for 30 min at 37 °C with 180 rpm shaking. Red blood cells were then removed by treatment with red blood cell lysis buffer (Beyotime, C3702) and the single-cell suspension was washed with RPMI medium containing 10% FBS (VivaCell) and filtered through a 74- pm nylon mesh. Cells were then counted and placed on ice for subsequent analysis.

[0219] 2. Flow cytometry sample preparation

[0220] After counting, cells in tissues were resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and all surface staining was blocked with anti-mouse CD16 / 32 antibody to prevent non-specific binding to Fc receptors. For surface staining, cells were stained with the designated antibodies according to the pre-set antibody panel at 4 °C in the dark for 30 min. For cytokine staining, cells were stimulated with cell stimulation cocktail (eBioscience) for 4 h before analysis. For intracellular staining, cells were first stained for surface markers and then fixed and permeabilized using the BD Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions, followed by staining with cytokine antibodies. For analysis of transcription factor Foxp3 expression, cells were first stained for surface markers and then pre-treated with Foxp3 / transcription factor staining buffer set (eBioscience) before staining with anti-Foxp3 antibody. Stained cells were washed with lx MACS buffer, data were acquired using a Cytoflex S flow cytometer (Beckman), and analyzed using FlowJo software (Tree Star).

[0221] 3. Real-time cytotoxicity assay experiment

[0222] Cytotoxicity of CTLs was determined by real-time cell analysis technology (RTCA), which uses the xCELLigence RTCA-MP instrument (ACEA Biosciences) to measure the number of adherent target cells that are not killed in real time. In the experiment, B16-OVA cells (1 x 105per well) were seeded in the wells of an E-Plate 16 (ACEA Biosciences) and incubated at 37 °C for 24 h to allow cell attachment. CTLs were then added to the wells at a 10: 1 effector-to-target ratio and incubated at 37 °C. The impedance signal was recorded every 15 min, and the number of live cells was calculated using the RTCA software (ACEA Biosciences). 4E-Plate 16 as target cells. After 12 hours, when B16-OVA cells were fully attached on E-Plate, CTLs (5 x 105cells per well) as effector cells were seeded in E-Plate 16 for killing. E-Plate 16 was placed in RTCA-MP instrument for 24 hours detection (37°C, 5% CO2). Cell index (CI) data of each group represented the mean value of three wells. 4 + / + The CI data of each group represented the mean value of three wells.

[0223] 4. RNA sequencing experiment

[0224] Primary CD8+T cells isolated from three pairs of 8-week-old male Sidt1 + / + and Sidt1 - / - mice were used for the experiment. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, US). Total RNA was treated by Berrygenomics (Beijing, China) and constructed mRNA library, then standard Illumina sequencing was performed on Novaseq 6000 system and more than 40 million paired-end 150 reads were obtained for each sample. Raw RNA sequencing reads were aligned to mouse genome (mm10, GRCm38) using STAR (v2.5.3a). Gene expression level and differential analysis were performed using edgeR (v3.29.2). Genes were considered significantly differentially expressed if showed >1.5-fold change in expression and FDR <0.05. Gene set analysis and enriched pathways were performed by online bioinformatics tool (metascape) and GSEA (v4.0.3). Volcano plots and pathway plots were generated using R package ‘ggplot2’.

[0225] 5. Ca 2+ Imaging experiment

[0226] Transfected HEK293 T cells were grown on poly-L-lysine-coated coverslips, incubated with 2 mM Fura-2 AM and 0.02% Pluronic F-127 for 30 min at 37°C in an isotonic solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES (adjusted to pH 7.4 with NaOH). The same solution without CaCl2was used as imaging buffer. After staining, cells were washed twice with imaging buffer and then transferred to the imaging chamber. Cells were treated with Thapsigargin (TG) to induce endoplasmic reticulum release of Ca 2+ CD8+T cells were attached on 48-well plates, then Ca2+ Imaging experiments. Ca 2+ Imaging system captures Ca 2+ Transients. Data collection and analysis were performed using MetaFluor software (Molecular Devices). Ratio measurements were performed by switching the excitation wavelength from 340 nm to 380 nm and quantifying the emission at 510 ± 40 nm.

[0227] 6. Experimental results

[0228] The results of Example 3 are shown in Figure 3. Specifically, Figure 3 shows that SIDT1 regulates the function of cytotoxic T lymphocytes (CTLs) by modulating the Ca 2+ levels in the cytoplasm of CD8+T cells. Figures 3a-3b It is shown that Sidt1 - / - CD8+T cells express higher CD69 under different stimulation times and different concentrations of anti-CD3 stimulation. Figures 3c-3d It is shown that Sidt1 - / - CD8+T cells show enhanced effector functions, including cytokine and granzyme production, and higher proliferation levels after stimulation. Figures 3e-3g It is shown that cytotoxic T cells with SIDT1 gene knockout have stronger killing ability and produce more interferon-γ when co-cultured with B16-OVA cells. Figures 3h-3i It is shown that CD8+T cells RNA sequencing and pathway enrichment analysis of Sidt1 gene knockout mice and their littermates find that the Ca 2+ homeostasis is affected. Figures 3j-3k It is shown that Sidt1 - / - T cells show enhanced Ca 2+ flux into the cells.

[0229] Therefore, the results of Example 3 show that SIDT1 knockout CD8+T cells have higher activation levels, stronger cytokine secretion ability, and stronger proliferation ability. And its killing function on tumors in vitro is also stronger. RNA sequencing results show that the Ca 2+ homeostasis of cells is affected, and subsequent experimental results also prove this. SIDT1 knockout CD8+T cells have higher intracellular Ca 2+ concentration, and adding Ca 2+ After transient stimulation, the intracellular calcium concentration is also higher. This shows that SIDT1 regulates the function of CTLs by affecting the Ca 2+ homeostasis of CD8+T cells.

[0230] Example 4. SIDT1 is a Ca2+ sensor specific to cytotoxic T lymphocytes 2+ Transporter

[0231] 1. Protein expression and purification

[0232] The DNA sequence of full-length human SIDT1 is publicly available at Uniprot database with accession number Q9NXL6-2. The cDNA was cloned into pCAG vector with a C-terminal Flag tag (DYKDDDDK). Site-directed mutations were generated by standard two-step PCR and verified by DNA sequencing. HEK293F cells (Sino Biological Inc.) were cultured at 37 °C, 5% CO2-supplemented SMM 293T-II medium (Sino Biological Inc.) and when the cell density reached 2.0 x 10 6Cells were transiently transfected with a mixture of hSIDT1 expression plasmid and polyethylenimine (PEIs) (Polysciences) at a 1 :3 ratio of DNA to PEI. Twelve hours post-transfection, 10 mM sodium butyrate (Sigma Aldrich) was added to the cell culture medium and the temperature was lowered to 30 °C to enhance protein expression. Approximately 72 hours post-transfection, cells were harvested by centrifugation at 3000 rpm for 10 min and the pellet was resuspended in lysis buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, supplemented with 1.3 mg / mL aprotinin, 5 mg / mL leupeptin, 0.7 mg / mL pepstatin and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich). The suspension was then added with 0.5% (w / v) lauryl maltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w / v) cholesterol hemisuccinate (CHS, Sigma Aldrich) and incubated at 4 °C for 2 hours. After centrifugation at 13000 rpm for 60 min, the supernatant was incubated with anti-FLAG M2 affinity gel (Sigma Aldrich) at 4 °C for 30 min. The resin was washed three times with wash buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.002% (w / v) LMNG and 0.0004% (w / v) CHS. Elution was performed using the wash buffer supplemented with 0.2 mg / mL FLAG peptide and the eluate was concentrated by an Amicon (Millipore) with a 10 kDa cutoff and further purified by size exclusion chromatography (Superose 6 increase 10 / 300 GL, GE Healthcare) in a buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.001% LMNG. The peak fractions were pooled and concentrated to approximately 5 mg / mL for cryo-EM sample preparation. Protein purification and sample handling were performed at 4 °C.

[0233] 2. Cryo-EM sample preparation and data collection

[0234] Four piL of purified human SIDTl protein sample was added to glow discharge treated carbon film grids (Quantifoil Au R1.2 / 1.3, 300 mesh) and plunge frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific) at 8 °C and 100% humidity by touching the grid for 3 seconds before plunging into the liquid ethane. The grid was transferred to a Titan Krios cryo-electron microscope (Thermo Fisher Scientific) operating at 300 kV. Micrographs stacks were automatically recorded in super-resolution mode using the EPU software (Thermo Fisher Scientific) with a nominal magnification of 81,000x and a defocus value between -1.5 and -2.3 pm using a Gatan Quantum energy filter followed by a K3 direct electron detector. The exposure time was 3.2 seconds for 32 frames per stack, for a total dose of The calibrated pixel size was

[0235] 3. Model building and refinement

[0236] The initial model was built by docking the Alphafold2 predicted hSIDTl model (AF-Q9NXL6-F1) into the 3.79 Angstrom cryo-EM map using UCSF Chimera. Then, model adjustment and de novo building were performed using Coot according to the cryo-EM density. Structure refinement was performed in real space by PHENIX with secondary structure and geometry constraints. Overfitting of the model was monitored by refining the model in one of two independent maps in the gold-standard refinement method and testing the refined model for adaptability to the other map.

[0237] 4. Electrophysiology

[0238] Patch-clamp recordings were performed using an Axopatch 200B patch-clamp amplifier (Molecular Devices) and a Digidata 1550B data acquisition system (Molecular Devices). Recording electrodes were made from borosilicate glass tubes using a P-1000 puller (Sutter Instrument). For whole-cell recordings, the electrode solution contained (in mM): 135 NMDG, 20 TEA, 10 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose, with or without 2 ATP-Mg (pH 7.2 adjusted with methanesulfonic acid). To test Ca 2+ and Ba 2+Transport capacity, 10 mM NMDG was replaced by 10 mM CaCl2or 10 mM BaCl2, respectively. The bath solution contained (in mM): 150 NMDG, 2 MgCl2, 10 HEPES, 10 glucose and 5 TEA (pH 7.2 adjusted with methanesulfonic acid). Cell membrane potential was held at -70 mV. Currents were recorded using a ramp protocol (from -100 mV to +100 mV in 500 ms, every 10 s). Liquid junction potentials were corrected online. Data were analyzed using Clampfit 10.7 (Molecular Devices) and OriginPro 2019 (OriginLab).

[0239] 5. Surface Plasmon Resonance (SPR) experiments

[0240] Surface Plasmon Resonance (SPR) experiments are an analytical technique used to detect metal cations (such as Ca 2+ , Ba 2+ , Zn 2+ , K + , Na + , etc.), ATP and SIDT1 inhibitor candidates. In this experiment, SPR experiments were performed on a Biacore 8K system (Cytiva) which is capable of performing experiments at 30 pL / min flow rate at 25 °C. Wild-type SIDT1 protein was immobilized on a Cytiva Series S CM5 sensor chip by amine coupling chemistry. Ligands at different concentrations were flowed over the chip surface in HBS-N buffer (Cytiva, BR100670) which contains 10 mM HEPES (pH 7.4), 150 mM NaCl. Data were analyzed by using Biacore Insight Evaluation Software version 3.0.12 which employs a steady-state affinity binding model.

[0241] 6. ATPase activity assay

[0242] ATPase activity of wild-type human SIDT1, mouse SIDT1 and variants was measured in 96-well plates at OD650 using Innova Biosciences ATPase colorimetric kit. To measure ATPase activity under different substrates, 3 micrograms of protein were added to the reaction buffer containing 25 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 10 mM MgCl2and 0.001% (w / v) LMNG. Then, each substrate was diluted into different concentrations and added to the mixture, incubated on ice for 30 minutes. After that, ATP was supplemented to the solution at a final concentration of 3 mM and reacted for 30 minutes at 37 °C.

[0243] 7. Experimental results

[0244] Results of Example 4 are shown in Figure 4. Specifically, Figure 4 shows that SIDT1 is a Ca 2+ transporter specific to cytotoxic T lymphocytes. Figures 4a-4d Electron density structure of SIDT1, dimer and monomer form of the protein, electrostatic potential of the protein surface are shown. Figures 4e-4i Whole-cell patch clamp experiments show that HEK293T cells overexpressing SIDT1 exhibit significantly larger outward currents than cells transfected with empty vector plasmid when Ca 2+ is added, and no current when Ba 2+ ions are added. Figures 4j-4k Surface plasmon resonance results further demonstrate that purified SIDT1 protein directly binds to Ca 2+ in vitro, and has a weaker binding ability to Ba 2+ . Figures 4l-4n Residues containing negative charges in the pore of SIDT1 structure are shown, and these sites play an important role in Ca 2+ transport. Figures 4o-4p SIDT1 requires ATP to transport Ca 2+ . Figures 4q-4r Purified SIDT1 protein directly binds and hydrolyzes ATP in vitro.

[0245] Therefore, Example 4 shows the SIDT1 structure solved by cryo-EM, and finds that it has a negative charge-accumulated pore region, which is consistent with the previous effect of Ca 2+ stability, and it is speculated that it can transport Ca 2+ . Subsequent patch clamp experiments prove this result, and it is found that SIDT1 can directly bind Ca 2+ and has ATPase activity, and has the ability to directly transport Ca 2+ . But it does not transport other cations.

[0246] Example 5. Relamorelin (TFA) enhances CD8+ T cell function to inhibit tumor growth by targeting SIDT1

[0247] 1. Isolation and activation of CD8+ T cells

[0248] MojoSort TM Mouse CD8 Unactivated CD8+ T cells were isolated using the Pan T Cell Isolation Kit (BioLegend). Cells were then stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies at the indicated concentrations for the indicated times. Cells were then stained to detect surface markers, fixed and permeabilized, and stained to detect intracellular cytokines.

[0249] 2. Virtual screening strategy

[0250] The crystal structure of human SIDT1 was used as the protein receptor. The protein was prepared using the Protein Preparation Wizard of Schrodinger® , resulting in an optimized and protonated structure that can be used for docking with Glide. The preparation process first removes water molecules that are more than 3.0 angstroms away from the protein. Then the conformation is repaired by filling in any missing residues. Finally, the protein structure is subjected to a restrained minimization using the OPLS_2005 force field with an RMSD tolerance of 0.30 angstroms. After preparation, the Receptor Grid Generation tool in Glide was used to generate a receptor grid box for ligand docking. The box was centered on the key residues (E555, S559, and D574) and extended 10 angstroms in all three dimensions. The MCE-Drug Repurposing Compound Library was used for virtual screening, containing 10247 compounds. All compounds were prepared using LigPrep in the "Virtual Screening Workflow" tool of Schrodinger® . Duplicate removal, generation of protomers using Epik at target pH 7.0 ± 2.0, removal of high-energy ionization / isomer states, and then retention of up to 4 stereoisomers and generation of 1 low-energy ring conformation were performed. After LigPrep, the compounds were docked to SIDT1 using the Glide HTVS, Glide Standard Precision (SP), and Glide Extra Precision (XP) methods in that order. Finally, the results of the Glide XP docking were analyzed, and a set of compounds were selected for further analysis.

[0251] 3. Relamorelin (TFA) affinity experiment with SIDT1 and patch clamp experiment

[0252] 4. Relamorelin (TFA) effect on CD8+ T cell effector function in vitro and in vivo

[0253] 5. Preparation of CAR-T lentivirus

[0254] The second generation of CD19-targeted CARs (chimeric antigen receptors) were composed of FMC63-scFv domain, CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta signaling domain, which were cloned into the third generation lentiviral expression vector pCDH-EF1 alpha-MCS vector backbone. Lentivirus was produced by transfecting 70-80% 293T cells to near confluence state using the transfection plasmids, pMDLg / pRRE, pRSV-REV, and pMD2.G, and polyethylenimine (PEI) transfection method. Virus-containing supernatants were collected at 48 and 72 hours post-transfection, centrifuged at 2000g for 10 minutes, and then filtered through a 0.45 pm PVDF filter. Virus was concentrated by ultracentrifugation at 25,000g for 4 hours at 4°C and resuspended with OptiVitro T Cell SF media and then stored at -80°C for future use.

[0255] 6. Preparation of CAR-T cells

[0256] Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donated by healthy volunteers by density gradient centrifugation. T lymphocytes were enriched from PBMCs using CD4+ and CD8+ T cell isolation kits from Miltenyi Biotech. T cells were then activated by CD3 and CD28 using T cell TransAct stimulation reagent from Miltenyi Biotech in OptiVitro T Cell SF media (ExCell Biotech) supplemented with 10 ng / mL IL-7 and 10 ng / mL IL-15 (Novoprotein) at a cell density of 10 cells / mL. After 24 hours of incubation, activated T cells were transduced with viral vectors encoding CD19-BBz CAR or EGFP. Transduced CAR-T or Mock-T cells were supplemented with fresh media every 2 to 3 days, which was supplemented with 10 ng / mL IL-7 and 10 ng / mL IL-15 for further cell expansion. 6

[0257] 7. Effect of Relamorelin (TFA) on CAR-T cells

[0258] ​The prepared CAR-T cells on the tenth day after activation were reactivated under CD3 and CD28 conditions and pretreated with 10 uM Relamorelin (TFA) or a control group for 12 h, and then co-incubated with Raji tumor cells. The cells secreting killing factors were detected by co-incubating for 4 h under the condition of blocking agents of brefeldin A and monensin, and then sampling for flow detection. The cells for detecting Raji killing were first labeled with CTV, and then the apoptosis of Raji was detected after co-incubation for 24 h.

[0259] 8. Experimental results

[0260] The results of Example 5 are shown in Figure 5. Specifically, Figure 5 shows that Relamorelin (TFA) enhances CD8+T cell function to inhibit tumor growth by targeting SIDT1. Figure 5a The candidate inhibitors after virtual screening are shown. Figures 5b-5c The effects of these candidate inhibitors on calcium flow inhibition and PD-1 expression of CD8+T cells overexpressing SIDT1 are shown, and it is found that Relamorelin (TFA) can significantly compensate for the calcium flow inhibition phenotype caused by overexpression of SIDT1, and can significantly promote PD-1 expression. Figure 5d The ability of Relamorelin (TFA) to directly bind to SIDT1 is shown. Figures 5e-5f In patch clamp experiments, Relamorelin (TFA) can compensate for the outward current caused by overexpression of SIDT1. Figures 5g-5h Relamorelin (TFA) significantly enhances the killing function of CD8+T cells in vitro. Figures 5i-5m Relamorelin (TFA) can also significantly enhance the function of CD8+T cells in vivo to inhibit tumor growth. Figures 5n-5p Relamorelin (TFA) in vitro treatment of CAR-T can significantly enhance the function of CAR-T to secrete cytokines and kill tumor cells.

[0261] In addition, the results of Example 5 show that 16 candidate compounds were found based on the virtual screening of the structure of SIDT1, and subsequent tests on the effects of calcium flow and CD8+ T cell expression of PD-1 showed that Relamorelin (TFA) can directly bind to SIDT1 and can significantly inhibit the intracellular calcium reduction caused by overexpression of SIDT1. In addition, Relamorelin (TFA) can also significantly enhance the effector function of CD8+ T cells in vitro and in vivo. Relamorelin (TFA) also has a significant enhancement effect on CAR-T therapy. These data show that Relamorelin (TFA) as a small molecule inhibitor has certain potential and advantages in the field of tumor immunotherapy.

[0262] All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the application herein disclosed in the drawing for which there is no intention of departing from the scope thereof, as defined by the appended claims.

Claims

1. Use of SIDT1 as an immune checkpoint.

2. Use according to claim 1, characterized in that, The use includes but is not limited to the development or preparation of a medicament for treating or preventing a SIDT1 -related disease, the development or preparation of a diagnostic reagent for a SIDT1 -related disease.

3. Use according to claim 2, characterized in that, The SIDT1 -related disease is a tumor with high expression of SIDT1; preferably, the tumor includes but is not limited to colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer; more preferably, the tumor is colorectal cancer or prostate cancer.

4. Use of SIDT1 as a prognostic marker for a SIDT1 -related disease.

5. Use of a SIDT1 inhibitor in the preparation of a medicament for preventing or treating a SIDT1 -related disease, or a diagnostic reagent for a SIDT1 -related disease.

6. An inhibitor of SIDT1.

7. A pharmaceutical composition comprising the following and a pharmaceutically acceptable excipient: 1) a SIDT1 inhibitor; or 2) an immune effector cell with a knock-out of SIDT1; or 3) a SIDT1 inhibitor and an immune effector cell without a knock-out of SIDT1; or 4) a SIDT1 inhibitor and an immune effector cell with a knock-out of SIDT1.

8. A combination of a SIDT1 inhibitor and other immune checkpoint inhibitors.

9. Use of the combination of a SIDT1 inhibitor and other immune checkpoint inhibitors according to claim 8 in the preparation of a medicament for a SIDT1 -related disease.

10. A method of enhancing or restoring the function of an immune effector cell, the method comprising the step of contacting a SIDT1 inhibitor with the immune effector cell; or, the step of knocking out SIDT1 of the immune effector cell.

11. Use of an immune effector cell with a knock-out of SIDT1 in the preparation of a medicament against a tumor.

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