Use of Cellular Immunotherapy

Abstract

The present invention provides a cancer treatment composition and a cancer treatment method for patients who have failed treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

Description

[Technical field]

[0001] The present invention belongs to the field of immunotherapy, and specifically relates to immune cell therapy that recognizes tumor antigens as targets, activates immune effector cells, and exerts antitumor effects. [Background technology]

[0002] Programmed death-1 (PD-1) is an immunosuppressive protein that negatively regulates TCR signals. PD-L1 is a ligand of PD-1 and is expressed in many types of tumors, such as ovarian cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, gastric cancer, melanoma, glioblastoma, non-small cell lung cancer, etc. (Callahan 2014 J Leukoc Biol 94(1):41-53). When PD-1 binds to PD-L1, it provides an inhibitory signal, induces apoptosis of T cells, and inhibits T cell activation and proliferation. Therefore, drugs targeting PD-1 or PD-L1 can exert antitumor effects through immune regulation. Currently, PD-1 antibodies or PD-L1 antibodies that are already on the market include Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, and Durvalumab. However, there are still many patients whose disease progresses after treatment with PD-1 antibodies or PD-L1 antibodies, resulting in treatment failure.

[0003] Cellular immunotherapy is a treatment method that uses the patient's own (or allogeneic) immune cells to repair tissues and organs. For example, CAR-T cell therapy modifies the patient's own (or allogeneic) T cells with chimeric antigen receptors to treat diseases such as cancer. As with cancer treatments with PD-1 or PD-L1 antibodies, both of these kill tumors through the immune system. Patients who have failed treatments with PD-1 or PD-L1 antibodies may be less suitable for tumor immunotherapy, and patients who are resistant to PD-1 or PD-L1 antibody treatments may be insensitive to other immunotherapies (e.g., CAR-T cell therapy). Summary of the Invention

[0004] The object of the present invention is to provide a composition and a method for treating cancer, in particular to provide a method and composition for treating cancer for patients who have failed anti-PD-1 antibody and / or anti-PD-L1 antibody treatment.

[0005] Specifically, the present invention relates to the following: 1. Use of a cell therapy product in the manufacture of a medicament for treating cancer in a patient who has failed a prior cancer treatment, wherein the prior treatment comprises treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

[0006] 2. The use of item 1, wherein the cell therapy product contains immune effector cells expressing an exogenous receptor.

[0007] 3. The use according to item 2, wherein the immune effector cells are selected from T cells, NK cells, NK T cells, mast cells, macrophages, dendritic cells, CIK cells, stem cell-derived immune effector cells, or combinations thereof.

[0008] 4. The use according to item 2, wherein the immune effector cells are derived from natural T cells and / or T cells induced by pluripotent stem cells.

[0009] 5. The use according to any one of items 2 to 4, wherein the immune effector cells are autologous / allogeneic T cells or primary T cells.

[0010] 6. The use according to any one of items 3 to 5, wherein the T cells comprise stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Treg), effector memory T cells (Tem), γδ T cells, αβ T cells, or a combination thereof.

[0011] 7. The use according to any one of items 2 to 6, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell fusion protein (TFP), a T cell-antigen coupler (TAC), or a combination thereof.

[0012] 8. The use according to any one of items 2 to 7, wherein the antigen-binding domain of the exogenous receptor specifically recognizes a tumor antigen.

[0013] 9. The use according to item 8, wherein the tumor antigen is selected from CD19, CD20, CD22, CD30, mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, or a combination thereof, preferably, the tumor antigen is selected from GPC3, CD19, BCMA, Claudin18.2, or a combination thereof.

[0014] 10. The use according to any one of items 1 to 9, wherein the cancer comprises a solid tumor and / or a blood tumor, preferably the solid tumor comprises a gastrointestinal tumor, more preferably the gastrointestinal tumor comprises gastric cancer / esophagogastric junction adenocarcinoma, gallbladder cancer, liver cancer, Krukenberg tumor, or a combination thereof.

[0015] 11. The use according to any one of items 1 to 10, wherein the cancer is selected from Krukenberg tumor, gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, liver cancer, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), and breast cancer.

[0016] 12. The use according to any one of items 1 to 11, wherein the anti-PD-1 antibody / PD-L1 antibody is selected from Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalimab, Sintilimab, Tislelizumab, Dimvelelimab (GLS-010), Tislelizumab, or a combination thereof.

[0017] 13. The use according to any one of items 1 to 12, wherein the preceding treatment comprises drug therapy, surgical treatment, radiation therapy or a combination thereof, and the drug therapy comprises administration of a chemical drug and / or a biological drug, preferably, the preceding treatment comprises administration of olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compounds, bevacizumab, regorafenib, anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib or a combination thereof.

[0018] 14. The use according to item 1, wherein the patient suffering from said cancer is treated by administering at least one cycle of the cell therapy product, preferably the patient suffering from said cancer is treated by administering 1 to 3 cycles of the cell therapy product.

[0019] 15. The chimeric antigen receptor (CAR), (i) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, CD3ζ, (ii) an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, and CD3ζ; (iii) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory signal domain of CD137, and CD3ζ, and / or (iv) The use according to any one of Items 7 to 14, comprising an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and CD3ζ.

[0020] 16. The dose of cells in the cell therapy product administered per cycle is approximately 2 x 10 9 Cells / kg, approximately 2 x 10 8 cells / kg or approximately 2 x 10 7 The dose of cells in cell therapy products should not exceed 1 x 10 cells / kg patient weight. 11 cells / patient, approximately 1 x 10 10 cells / patient, approximately 5 x 10 9 cells / patient, approximately 2 x 10 9 cells / patient or approximately 1 x 10 9 16. The use according to item 14 or 15, wherein the number of cells per patient is not more than 10.

[0021] 17. The dose of cells in the cell therapy product administered per cycle is approximately 1 x 10 5 cells / kg patient weight ~ approx. 2 x 10 7 cells / kg patient weight, or approximately 1 x 10 6 cells / kg patient weight ~ approx. 2 x 10 7 cells / kg patient weight, or The dose of cells in the cell therapy product administered per cycle is approximately 1 x 10 7 ~ approx. 5 x 10 cells 9 cells / patient, approximately 1 x 10 7 ~ approx. 2 x 10 cells 9 cells / patient, or approximately 1 x 10 7 ~ approx. 1 x 10 cells 9The dose of cells in a cell therapy product administered per cycle is approximately 1 x 10 cells / patient. 8 ~ approx. 5 x 10 cells 9 cells / patient, approximately 1 x 10 8 ~ approx. 2 x 10 cells 9 cells / patient, or approximately 1 x 10 8 ~ approx. 1 x 10 cells 9 The dose of cells in a cell therapy product administered per cycle is approximately 2.5×10 cells / patient. 8 ~ approx. 5 x 10 cells 8 Item 17. The use according to item 16, wherein the cell number is 1 cell / patient.

[0022] 18. The use according to any one of claims 14 to 17, wherein a pretreatment is performed prior to administration of the cell therapy product in each cycle, the pretreatment comprising administering to the patient a chemical drug, a biological drug, radiation therapy or a combination thereof.

[0023] 19. The use according to item 18, wherein the pretreatment is performed 1 to 8 days prior to administration of the cell therapy product, preferably 2 to 6 days prior to administration of the cell therapy product, preferably with no more than 4 days of continuous use of chemical drugs, biological drugs, radiation therapy or combinations thereof.

[0024] 20. The use according to item 18 or 19, wherein the chemical drug is any one or at least two selected from cyclophosphamide, fludarabine, a tubulin inhibitor, and a pyrimidine antitumor drug, or the chemical drug comprises cyclophosphamide and fludarabine, or the chemical drug comprises cyclophosphamide, fludarabine, and a tubulin inhibitor.

[0025] 21. The use according to item 20, wherein the tubulin inhibitor is a taxane compound, preferably the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably the taxane compound is albumin-bound paclitaxel.

[0026] 22. The dosage of fludarabine is approximately 10-50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or approximately 25 mg / m 2 / day, or about 30-60 mg / day, or about 30-50 mg / day, or about 35-45 mg / day; The dose of cyclophosphamide is approximately 200-400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day, or about 300-700 mg / day, or about 300-550 mg / day, or about 300-500 mg / day; 22. The use according to item 20 or 21, wherein the dosage of the taxane compound is about 300 mg / day or less, about 200 mg / day or less, or about 90 to 120 mg / day.

[0027] 23. The use according to any one of items 20 to 22, wherein the cyclophosphamide is administered 2 to 3 times, or the fludarabine is administered 1 to 2 times, or the taxane compound is administered once.

[0028] 24. The antigen-binding domain comprises: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6, or HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21, or It has HCDR1 shown in SEQ ID NO: 27, HCDR2 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, and LCDR3 shown in SEQ ID NO: 32, or the antigen-binding domain is A heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 9, or A heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 23, or A heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 34, Or the use according to any one of items 8 to 15, wherein the antigen-binding domain comprises the sequence shown in SEQ ID NO: 14, 24, 35, or 37.

[0029] 25. The use according to any one of Items 7 to 24, wherein the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, or 52, or comprises a polypeptide comprising a sequence as shown in any one of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67 linked in sequence to a sequence as shown in SEQ ID NO: 80, 81, or 82, respectively, or comprises a polypeptide comprising a sequence as shown in any one of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79 linked in sequence to a sequence as shown in SEQ ID NO: 80, 81, or 82, respectively.

[0030] 26. The use according to any one of clauses 14 to 25, wherein the patient is assessed for serum levels of cytokines indicative of CRS, cytokines indicative of neurotoxicity, indicators of tumor burden, and / or factors indicative of a host anti-CAR immune response prior to administration of the cell therapy product in each cycle.

[0031] 27. The use according to any one of paragraphs 1 to 26, wherein after treatment with the cell therapy product, the patient does not show severe CRS or does not show neurotoxicity exceeding grade 3.

[0032] 28. The use according to any one of clauses 1 to 27, wherein at least a portion, preferably at least 40%, more preferably at least 50%, of patients who have failed cancer treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.

[0033] 29. The use according to clause 28, wherein the response is an ORR greater than 40%, preferably greater than 50%.

[0034] 30. A method of treating cancer in a patient who has failed prior cancer treatment with a cell therapy product, wherein the prior treatment comprises treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

[0035] 31. The method of claim 30, wherein the cell therapy product contains immune effector cells expressing an exogenous receptor.

[0036] 32. The method according to item 31, wherein the immune effector cells are selected from T cells, NK cells, NK T cells, mast cells, macrophages, dendritic cells, CIK cells, stem cell-derived immune effector cells, or combinations thereof.

[0037] 33. The method of claim 31, wherein the immune effector cells are derived from natural T cells and / or T cells induced by pluripotent stem cells.

[0038] 34. The method according to any one of items 31 to 33, wherein the immune effector cells are autologous / allogeneic T cells or primary T cells.

[0039] 35. The method according to any one of items 32 to 34, wherein the T cells comprise stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Treg), effector memory T cells (Tem), γδ T cells, αβ T cells, or a combination thereof.

[0040] 36. The method according to any one of items 31 to 35, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell fusion protein (TFP), a T cell-antigen coupler (TAC), or a combination thereof.

[0041] 37. The method according to any one of items 31 to 36, wherein the antigen-binding domain of the exogenous receptor specifically recognizes a tumor antigen.

[0042] 38. The method according to item 37, wherein the tumor antigen is selected from CD19, CD20, CD22, CD30, mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, or a combination thereof, preferably, the tumor antigen is selected from GPC3, CD19, BCMA, Claudin18.2, or a combination thereof.

[0043] 39. The method according to any one of items 30 to 38, wherein the cancer comprises a solid tumor and / or a blood tumor, preferably the solid tumor comprises a gastrointestinal tumor, more preferably the gastrointestinal tumor comprises gastric cancer / esophagogastric junction adenocarcinoma, gallbladder cancer, liver cancer, Krukenberg tumor, or a combination thereof.

[0044] 40. The method according to any one of items 30 to 39, wherein the cancer is selected from Krukenberg tumor, gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, liver cancer, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), and breast cancer.

[0045] 41. The method according to any one of items 30 to 40, wherein the anti-PD-1 antibody / PD-L1 antibody is selected from nivolumab, pembrolizumab, cemiplimab, camrelizumab, toripalimab, sintilimab, tislelizumab, dimverelimab (GLS-010), tislelizumab, or a combination thereof.

[0046] 42. The method according to any one of items 30 to 41, wherein the preceding treatment comprises drug therapy, surgical treatment, radiation therapy or a combination thereof, and the drug therapy comprises administration of a chemical drug and / or a biological drug, preferably, the preceding treatment comprises administration of olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compounds, bevacizumab, regorafenib, anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib or a combination thereof.

[0047] 43. The method according to claim 30, wherein the patient suffering from said cancer is treated by administering at least one cycle of the cell therapy product, preferably the patient suffering from said cancer is treated by administering 1 to 3 cycles of the cell therapy product.

[0048] 44. The chimeric antigen receptor (CAR), (i) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, CD3ζ, (ii) an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, and CD3ζ; (iii) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory signal domain of CD137, and CD3ζ, and / or (iv) The method according to any one of items 36 to 43, comprising an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and CD3ζ.

[0049] 45. The dose of cells in the cell therapy product administered per cycle is approximately 2 × 10 9 Cells / kg, approximately 2 x 10 8 cells / kg or approximately 2 x 10 7 The dose of cells in cell therapy products should not exceed 1 x 10 cells / kg patient weight. 11cells / patient, approximately 1 x 10 10 cells / patient, approximately 5 x 10 9 cells / patient, approximately 2 x 10 9 cells / patient or approximately 1 x 10 9 45. The method of claim 43 or 44, wherein the number of cells per patient is not more than 10.

[0050] 46. ​​The dose of cells in a cell therapy product administered per cycle is approximately 1 × 10 5 cells / kg patient weight ~ approx. 2 x 10 7 cells / kg patient weight, or approximately 1 x 10 6 cells / kg patient weight ~ approx. 2 x 10 7 cells / kg patient weight, or The dose of cells in the cell therapy product administered per cycle is approximately 1 x 10 7 ~ approx. 5 x 10 cells 9 cells / patient, approximately 1 x 10 7 ~ approx. 2 x 10 cells 9 cells / patient, or approximately 1 x 10 7 ~ approx. 1 x 10 cells 9 The dose of cells in a cell therapy product administered per cycle is approximately 1 x 10 cells / patient. 8 ~ approx. 5 x 10 cells 9 cells / patient, approximately 1 x 10 8 ~ approx. 2 x 10 cells 9 cells / patient, or approximately 1 x 10 8 ~ approx. 1 x 10 cells 9 The dose of cells in a cell therapy product administered per cycle is approximately 2.5×10 cells / patient. 8 ~ approx. 5 x 10 cells 8 46. ​​The method of claim 45, wherein the number of cells per patient is 1.

[0051] 47. The method of any one of paragraphs 43 to 46, wherein a pretreatment is performed prior to administration of the cell therapy product in each cycle, the pretreatment comprising administering to the patient a chemical drug, a biological drug, radiation therapy, or a combination thereof.

[0052] 48. The method according to paragraph 47, wherein the pretreatment is performed 1 to 8 days prior to administration of the cell therapy product, preferably 2 to 6 days prior to administration of the cell therapy product, preferably for no more than 4 days of continuous use of chemical drugs, biological drugs, radiation therapy or combinations thereof.

[0053] 49. The method according to item 47 or 48, wherein the chemical drug is any one or at least two selected from the group consisting of cyclophosphamide, fludarabine, a tubulin inhibitor, and a pyrimidine antitumor drug, or the chemical drug comprises cyclophosphamide and fludarabine, or the chemical drug comprises cyclophosphamide, fludarabine, and a tubulin inhibitor.

[0054] 50. The method according to item 49, wherein the tubulin inhibitor is a taxane compound, preferably the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably the taxane compound is albumin-bound paclitaxel.

[0055] 51. The dosage of fludarabine is approximately 10-50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or approximately 25 mg / m 2 / day, or about 30-60 mg / day, or about 30-50 mg / day, or about 35-45 mg / day; The dose of cyclophosphamide is approximately 200-400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day, or about 300-700 mg / day, or about 300-550 mg / day, or about 300-500 mg / day; Item 51. The method according to item 49 or 50, wherein the dosage of the taxane compound is about 300 mg / day or less, about 200 mg / day or less, or about 90 to 120 mg / day.

[0056] 52. The method according to any one of items 49 to 51, wherein the cyclophosphamide is administered 2 to 3 times, or the fludarabine is administered 1 to 2 times, or the taxane compound is administered once.

[0057] 53. The antigen-binding domain comprises: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6, or HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21, or It has HCDR1 shown in SEQ ID NO: 27, HCDR2 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, and LCDR3 shown in SEQ ID NO: 32, or the antigen-binding domain is A heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 9, or A heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 23, or A heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 34, 45. The method according to any one of items 37 to 44, wherein the antigen-binding domain comprises the sequence shown in SEQ ID NO: 14, 24, 35, or 37.

[0058] 54. The method according to any one of items 36 to 53, wherein the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, and 52; or comprises a polypeptide comprising a sequence as shown in any one of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67 linked in sequence to a sequence as shown in SEQ ID NO: 80, 81, or 82, respectively; or comprises a polypeptide comprising a sequence as shown in any one of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79 linked in sequence to a sequence as shown in SEQ ID NO: 80, 81, or 82, respectively.

[0059] 55. The method of any one of paragraphs 43 to 54, wherein the patient is assessed for serum levels of cytokines indicative of CRS, cytokines indicative of neurotoxicity, indicators of tumor burden, and / or factors indicative of a host anti-CAR immune response prior to administration of the cell therapy product in each cycle.

[0060] 56. The method according to any one of paragraphs 30 to 55, wherein after treatment with the cell therapy product, the patient does not show severe CRS or does not show neurotoxicity exceeding grade 3.

[0061] 57. The method according to any one of clauses 30 to 56, wherein at least a portion, preferably at least 40%, more preferably at least 50%, of patients who have failed cancer treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.

[0062] 58. The method of clause 57, wherein the response is an ORR greater than 40%, preferably greater than 50%.

[0063] The technical solutions provided by the present invention to achieve the above objectives are as follows: In a first aspect of the invention there is provided the use of a cell therapy product for the treatment of cancer after failure of prior treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0064] In a preferred embodiment, a proportion of patients who have failed prior treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.

[0065] In a preferred embodiment, at least 40% of patients who have failed prior treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.

[0066] In a preferred embodiment, at least 50% of patients who have failed prior treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.

[0067] In a preferred embodiment, the response is an ORR greater than 40%, preferably greater than 50%.

[0068] In a preferred embodiment, the preceding treatment also includes a chemical or biological drug, preferably, the chemical or biological drug is olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compounds, bevacizumab, or regorafenib.

[0069] In a preferred embodiment, the anti-PD-1 antibody is selected from Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalimab, Sintilimab, Tislelizumab, Gimverelimab (GLS-010), and Tislelizumab, and preferably, the anti-PD-1 antibody is selected from Nivolumab, Pembrolizumab, Camrelizumab, Toripalimab, Sintilimab, and Tislelizumab.

[0070] In a preferred embodiment, the anti-PD-L1 antibody is selected from Atezolizumab, Durvalumab, Avelumab, and Sugemalimab (CS1001).

[0071] In a preferred embodiment, the cell therapy product comprises immune effector cells expressing an exogenous receptor, preferably the immune effector cells are selected from T cells, NK cells, NK T cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.

[0072] In a preferred embodiment, said immune effector cells are selected from T cells and said exogenous receptors are selected from chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell fusion proteins (TFPs) and T cell-antigen couplers (TACs).

[0073] In a preferred embodiment, the exogenous receptor is selected from a chimeric antigen receptor (CAR).

[0074] In a preferred embodiment, said immune effector cells are selected from T cells, NK cells, and NK T cells.

[0075] In a preferred embodiment, the cell therapy product specifically recognizes a tumor antigen, preferably the tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, more preferably the tumor antigen is selected from CD19, BCMA, Claudin18.2.

[0076] In a preferred embodiment, the cancer is selected from gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), and breast cancer.

[0077] In a preferred embodiment, the cancer is selected from gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), urothelial carcinoma (UC), liver cancer, and melanoma.

[0078] In a preferred embodiment, the patient is treated with at least one cycle of the cell therapy product, preferably the patient is treated with 1 to 3 cycles of the cell therapy product.

[0079] In a preferred embodiment, the dose of cells in the cell therapy product administered per cycle is about 2×10 9 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 11 Preferably, the dose of cells in the cell therapy product administered per cycle is about 2×10 8 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 10 More preferably, the dose of cells in the cell therapy product administered per cycle is about 2×10 7 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 9 Not to exceed 10 cells / person.

[0080] In a preferred embodiment, the dose of cells in the cell therapy product administered per cycle is about 1×10 5 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of examinee's body weight, or approximately 1 x 10 6 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of examinee's body weight.

[0081] In a preferred embodiment, the dose of cells in the cell therapy product administered per cycle is greater than or equal to 1×10 7 cells ~ 1×10 9 cells, preferably 1 x 10 8 cells ~ 1×10 9 cells, more preferably 2.5×108 cells ~5×10 8 Each cell is a single cell.

[0082] In a preferred embodiment, subsequent cycles of treatment are administered after the cellular products of the previous cycle are no longer detectable in the body following treatment.

[0083] In a preferred embodiment, the subsequent cycle of cell therapy product is administered 30 days after completion of administration of the previous cycle of cell product.

[0084] In a preferred embodiment, the dose of cell product administered in the subsequent cycle is sufficient to stabilize or reduce the cell number of the recipient's tumor burden.

[0085] In a preferred embodiment, the doses of cell product administered in said different cycles are different or the same.

[0086] In a preferred embodiment, each cycle of the cell therapy product is administered 1-5 times, preferably 1-3 times.

[0087] In a preferred embodiment, all administration of each cycle of cell therapy product is completed within 5 days, and preferably, all administration is completed within 3 days.

[0088] In a preferred embodiment, each infusion of the cell therapy product is completed within 1 hour, preferably within 30 minutes, and more preferably within 4-30 minutes.

[0089] In a preferred embodiment, at the time of subsequent cycle treatment, the examinee has any of the following characteristics: (i) The peak levels in the test subject's serum of cytokines associated with cytokine release syndrome (CRS) are less than the peak levels following administration of the cell therapy product in the previous cycle. (ii) No evidence of grade 3 or higher neurotoxicity. (iii) the CRS level is reduced compared to the CRS level after administration of the previous cycle of the cell therapy product; (iv) the test taker does not demonstrate a detectable humoral or cell-mediated immune response to the cell therapy product.

[0090] In a preferred example, in (i) above, the cytokine level is reduced by at least 50%, preferably by at least 20%, and more preferably by at least 5%, compared to the peak cytokine level after administration of the previous cycle of the cell therapy product.

[0091] In a preferred embodiment, the CRS level is equivalent to the CRS level prior to administration of the previous cycle of the cell therapy product.

[0092] In a preferred embodiment, a pretreatment is administered prior to administration of the cell therapy product in each cycle, the pretreatment including administering chemotherapeutic and / or radiation therapy to the subject.

[0093] In a preferred embodiment, the radiation therapy comprises systemic radiation therapy or localized radiation therapy.

[0094] In a preferred embodiment, the pretreatment is administered 1-8 days prior to administration of the cell therapy product, preferably 2-6 days prior to administration of the cell therapy product.

[0095] In a preferred embodiment, the chemical drug is any one or at least two selected from the group consisting of cyclophosphamide, fludarabine, a tubulin inhibitor, and a pyrimidine antitumor drug.

[0096] In a preferred embodiment, the chemical drugs are cyclophosphamide and fludarabine, or cyclophosphamide, fludarabine and a tubulin inhibitor.

[0097] In a preferred example, the tubulin inhibitor is a taxane compound, preferably the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably the taxane compound is albumin-bound paclitaxel.

[0098] In a preferred embodiment, the pyrimidine antitumor drug is selected from 5-fluorouracil, tegadiful, carmofur, doxifluridine, and capecitabine.

[0099] In a preferred embodiment, the dosage of fludarabine is about 10 to 50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or about 25 mg / m 2 / day.

[0100] In a preferred embodiment, the dosage of fludarabine is about 30 to 60 mg / day, or about 30 to 50 mg / m 2 / day.

[0101] In a preferred embodiment, the dose of cyclophosphamide is about 200 to 400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day.

[0102] In a preferred example, the dosage of cyclophosphamide is about 300 to 700 mg / day, or about 300 to 550 mg / day, or about 300 to 500 mg / day.

[0103] In a preferred example, the dosage of the taxane compound is about 300 mg / day or less, or about 200 mg / day or less, and preferably, the dosage of the taxane compound is about 90 to 120 mg / day.

[0104] In a preferred embodiment, consecutive use of each chemical agent does not exceed four days.

[0105] In a preferred example, the cyclophosphamide is administered 2 to 3 times, or the fludarabine is administered 1 to 2 times.

[0106] In a preferred embodiment, the taxane compound is administered once.

[0107] In a preferred example, the exogenous receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen-binding domain comprises an antibody or a fragment thereof that specifically recognizes a tumor antigen, with the proviso that the antibody or the fragment thereof is HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6, or HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18, LCDR1 as set forth in SEQ ID NO: 19, LCDR2 as set forth in SEQ ID NO: 20, LCDR3 as set forth in SEQ ID NO: 21, or It has HCDR1 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:28, HCDR3 shown in SEQ ID NO:29, LCDR1 shown in SEQ ID NO:30, LCDR2 shown in SEQ ID NO:31, and LCDR3 shown in SEQ ID NO:32.

[0108] Preferably, the antibody or fragment thereof comprises: A heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 9, or A heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 23, or It has a heavy chain variable region shown in SEQ ID NO:33 and a light chain variable region shown in SEQ ID NO:34.

[0109] In a preferred embodiment, the extracellular antigen-binding domain of the exogenous receptor comprises the sequence shown in SEQ ID NO: 14, 24, 35, or 37.

[0110] In a preferred example, the chimeric antigen receptor has an amino acid sequence set forth in any one of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, and 52.

[0111] In a preferred embodiment, the subject has not undergone cell therapy prior to administration of the cell therapy product.

[0112] In a preferred embodiment, prior to administration of the cell therapy product, the subject may have also undergone medical therapy, surgical therapy, radiation therapy, or any combination thereof.

[0113] In a preferred embodiment, the medical therapy comprises a chemical or biological drug therapy.

[0114] In a preferred embodiment, the chemotherapy comprises anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib.

[0115] In a preferred embodiment, prior to administration of the cell therapy product in each cycle, the subject is assessed for serum levels of cytokines indicative of CRS, cytokines indicative of neurotoxicity, indicators indicative of tumor burden, and / or factors indicative of a host anti-CAR immune response.

[0116] In a preferred embodiment, the indicator of tumor burden is the total number of tumor cells in the test subject, or the total number of tumor cells in an organ of the test subject, or the total number of tumor cells in a tissue of the test subject, or tumor mass or volume, or the extent of tumor metastasis, or tumor quantity.

[0117] In a preferred embodiment, the results of said assessment determine the dosage of the cell therapy product to be administered.

[0118] In a preferred embodiment, after treatment with the cell therapy product, the subject does not experience severe CRS or neurotoxicity above grade 3.

[0119] In a preferred embodiment, after administration of the cell therapy product, the cell therapy product proliferates within the subject.

[0120] In a second aspect of the present invention, there is also provided a method of treating a tumour with exogenous receptor modified immune effector cells, said method comprising administering a pre-treatment agent to a tumour patient prior to administering the immune effector cells, said pre-treatment agent comprising cyclophosphamide, fludarabine and a tubulin inhibitor.

[0121] In a preferred embodiment, the dosage of fludarabine is about 10 to 50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or about 25 mg / m 2 / day.

[0122] In a preferred embodiment, the dosage of fludarabine is about 30-60 mg / day, or about 30-50 mg / m 2 / day.

[0123] In a preferred embodiment, the dose of cyclophosphamide is about 200 to 400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day.

[0124] In a preferred embodiment, the dosage of cyclophosphamide is about 300 to 700 mg / day, or about 300 to 550 mg / day, or about 300 to 500 mg / day.

[0125] In a preferred embodiment, the dosage of the taxane compound is about 300 mg / day or less, or about 200 mg / day or less, preferably, the dosage of the taxane compound is about 90-120 mg / day.

[0126] In a preferred embodiment, consecutive use of each prior treatment agent does not exceed four days.

[0127] In a preferred embodiment, the cyclophosphamide is administered 2 to 3 times, In a preferred embodiment, the fludarabine is administered once or twice.

[0128] In a preferred embodiment, the tubulin inhibitor is administered once.

[0129] In a preferred embodiment, the tubulin inhibitor is a taxane compound, preferably the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably the taxane compound is albumin-bound paclitaxel.

[0130] In a preferred embodiment, the exogenous receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen-binding domain comprises an antibody or a fragment thereof that specifically recognizes a tumor antigen, with the proviso that the antibody or fragment thereof is HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6, or HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18, LCDR1 as set forth in SEQ ID NO: 19, LCDR2 as set forth in SEQ ID NO: 20, LCDR3 as set forth in SEQ ID NO: 21, or It has HCDR1 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:28, HCDR3 shown in SEQ ID NO:29, LCDR1 shown in SEQ ID NO:30, LCDR2 shown in SEQ ID NO:31, and LCDR3 shown in SEQ ID NO:32.

[0131] Preferably, the antibody or fragment thereof comprises: A heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 9, or A heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 23, or It has a heavy chain variable region shown in SEQ ID NO:33 and a light chain variable region shown in SEQ ID NO:34.

[0132] In a preferred embodiment, the extracellular antigen-binding domain of the exogenous receptor comprises the sequence set forth in SEQ ID NO: 14, 24, 35, or 37.

[0133] In a preferred embodiment, said immune effector cells are selected from T cells, NK cells, NK T cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.

[0134] In a preferred embodiment, said immune effector cells are selected from T cells and said exogenous receptors are selected from chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell fusion proteins (TFPs) and T cell-antigen couplers (TACs).

[0135] In a preferred embodiment, the exogenous receptor is selected from a chimeric antigen receptor (CAR), preferably wherein the chimeric antigen receptor comprises an amino acid sequence as set forth in any of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, 52.

[0136] In a preferred embodiment, said immune effector cells are selected from T cells, NK cells, and NK T cells.

[0137] In a preferred embodiment, the immune effector cells modified by said exogenous receptor specifically recognize a tumor antigen, preferably said tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, more preferably said tumor antigen is selected from CD19, BCMA, Claudin18.2.

[0138] In a preferred embodiment, the tumor is selected from gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), and breast cancer.

[0139] In a preferred embodiment, the tumor is selected from gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), urothelial carcinoma (UC), liver cancer, and melanoma.

[0140] In a preferred embodiment, the subject has not undergone cell therapy prior to administration of the immune effector cells.

[0141] In a preferred embodiment, prior to administration of the immune effector cells, the subject may have also undergone drug therapy, surgical therapy, radiation therapy, or any combination thereof.

[0142] In a preferred embodiment, the drug therapy comprises a chemical or biological drug therapy, preferably, the chemical drug therapy comprises anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib.

[0143] In a preferred embodiment, the dose of cells in the cell therapy product administered per cycle is about 2×10 9 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 11 Preferably, the dose of immune effector cells administered per cycle is about 2×10 8 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 10 More preferably, the dose of immune effector cells administered per cycle is no more than about 2×10 cells / person. 7Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 9 Not to exceed 10 cells / person.

[0144] In a preferred embodiment, the dose of immune effector cells administered per cycle is about 1×10 5 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of subject's body weight, or approximately 1 x 10 6 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of subject body weight or the dose of immune effector cells administered per cycle is 1 x 10 7 cells ~ 1×10 9 cells, preferably 1 x 10 8 cells ~ 1×10 9 cells, more preferably 2.5×10 8 cells ~5×10 8 Each cell is a single cell.

[0145] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (for example, in the examples) can be combined with each other to form new or preferred technical solutions, which will not be described in detail here due to space limitations. [Brief description of the drawings]

[0146] [Figure 1] FIG. 1 is a waterfall plot of optimal change from baseline in tumor burden. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0147] Through extensive and in-depth research, the inventors have unexpectedly found that when anti-PD-1 and / or anti-PD-L1 antibodies are used in prior treatment for cancer patients and the treatment fails (disease progression PD according to RECIST 1.1), the efficacy of cell therapy products is not affected, and at least 40% of patients still respond to cell therapy products. For example, in the case of solid tumors, patients with gastric cancer / esophagogastric junction adenocarcinoma, Krukenberg tumor, liver cancer, and gallbladder cancer respond to cell therapy products (e.g., CAR-T cells) after anti-PD-1 and / or anti-PD-L1 antibody treatment fails. The disease control rate of gastric cancer / esophagogastric junction adenocarcinoma patients who have failed anti-PD-1 and / or anti-PD-L1 antibody treatment after treatment with CAR-T cells reaches about 79.2%. In liver cancer patients who had failed anti-PD-1 antibody and / or anti-PD-L1 antibody treatment, the disease control rate reached approximately 54.5% after treatment with CAR-T cells.

[0148] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or specific terms used herein have the same meaning as commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are further limited in order to facilitate explanation and / or reference in this specification, and if such further limitations are included in this specification, it is not intended to be understood that there is a substantial difference from the common understanding in the art.

[0149] All publications, including patent documents, journal articles, and databases, referred to in this specification are incorporated herein by reference in their entirety, and to the extent that the definitions set forth herein differ or may not coincide with those set forth in the patents, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall control.

[0150] In this application, the claimed subject matter is presented in a range format, and it should be understood that the description in range format is merely for convenience and brevity, and should not be taken as a strict limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered as if all possible subranges and single numerical values ​​within the range have already been specifically disclosed. For example, where an upper limit and a lower limit of a range are provided, every intermediate value between the upper limit and the lower limit of the range is included in the claimed subject matter, and the upper and lower limits of the range also belong to the scope of the claimed subject matter.

[0151] Any concentration range, percentage range, proportion range, or integer range described herein should be understood to include every integer and, in some cases, fractional values ​​thereof (e.g., tenths and hundredths of an integer) within the range, unless otherwise specified.

[0152] The term "about" as used herein refers to the normal error range of each value that is familiar to those skilled in the art. When a value or parameter is referred to herein with "about", it includes (and describes) an embodiment for the value or parameter itself. For example, a statement of "about X" includes a statement for "X". For example, "about" may mean less than or greater than 1 according to the practical standard deviation in the art. Or "about" may mean up to a 20% range, or up to a 10% range, or up to a 5% range, or up to a 1% range. For example, about 5 mg may include any value between 4.5 mg and 5.5 mg. When a specific value or composition is provided in the application documents and claims, unless otherwise specified, "about" should be assumed to be within the acceptable error range of the specific value or composition.

[0153] In this patent, when describing an amino acid or nucleic acid sequence, "comprising" a particular sequence should be understood to cover variants of the particular sequence. In this patent, the amino acid or nucleic acid sequence comprises a particular sequence, which means that the amino acid or nucleic acid sequence has more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the particular sequence. Sequence identity may be measured by sequence analysis software (e.g., programs such as BLAST, BESTFIT, GAP, PILEUP / PRETTYBOX, etc.). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the group of glycine and alanine, valine, isoleucine and leucine, aspartic acid, glutamic acid, asparagine and glutamine, serine and threonine, lysine and arginine, and phenylalanine and tyrosine. An exemplary method for determining the degree of identity may utilize the BLAST program, with a probability score between e-3 and e-100 indicating closely related sequences.

[0154] As used herein, "failure of prior treatment with an anti-PD-1 antibody and / or anti-PD-L1 antibody" means that, at the time of prior treatment, the patient has previously received PD-1 and / or PD-L1 antibody treatment, or has previously been treated with a therapy that includes a PD-1 and / or PD-L1 antibody, and the prior treatment has failed to control disease progression (e.g., the disease has progressed to progressive disease (PD) per RECIST 1.1 in solid tumors).

[0155] As used herein, a "therapy comprising a PD-1 and / or PD-L1 antibody" refers to a treatment in which, in addition to the use of a PD-1 and / or PD-L1 antibody, other drugs or therapies are also used according to the needs of the treatment, such as, for example, a treatment in which, in addition to the use of a PD-1 and / or PD-L1 antibody, radiation therapy, stem cell transplantation, etc. are also used, or a treatment in which, in addition to the use of a PD-1 and / or PD-L1 antibody, other chemical or biological drugs are also used, such as, for example, olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compounds, bevacizumab, etc., or combinations thereof.

[0156] The treatment of tumor patients is a very long process. The so-called first-line, second-line, third-line, and late-line (including fourth-line, fifth-line, etc.) treatments actually refer to the order of selection and use of tumor treatment plans. First-line treatment generally refers to the initial systemic chemical drug and / or biologic drug treatment for tumor patients who cannot be surgically removed when a tumor is diagnosed, and the initial systemic chemical drug and / or biologic drug treatment for tumor patients who can be surgically removed when the tumor recurs after surgery (with or without adjuvant therapy). Second-line treatment generally refers to the need to change to a different treatment plan after the first-line treatment has failed (including cases where the first-line treatment is effective but the patient experiences tumor progression again). Third-line treatment generally refers to changing to another treatment plan again after the second-line treatment has failed. Most tumors generally have fewer selectable drugs and effective treatment plans when they reach the third line.

[0157] PD1 and / or PD-L1 antibody therapy has already been approved for different lines of treatment depending on the tumor. Currently, in clinical practice, patients with PD-L1 positive or negative tumors may be treated with PD1 and / or PD-L1 antibodies, and the first, second, third, or late lines (fourth, fifth, etc.) of tumor treatment are all in clinical use with PD1 and / or PD-L1 antibodies alone or in combination with other drugs.

[0158] In the conventional gastric cancer treatment plan, when chemotherapy drugs (taxanes, irinotecan, etc.), targeted drugs (apatinib) and / or immune checkpoint inhibitors (nivolumab) are used, the objective response rate is about 2-11.2%, the mPFS is about 2-3 months, and the mOS is about 6 months. In the present invention, a cell therapy product (exemplarily, CAR-T cells) is used in the second-line, third-line or late-line treatment for gastric cancer / esophagogastric junction adenocarcinoma patients who have failed PD-1 and / or PD-L1 antibody treatment, and a higher response rate of about 50% is shown, the disease control rate is about 79.2%, the progression-free survival and overall survival are prolonged, and the patients have obtained significant clinical benefit. In 12 patients who achieved PR after CAR-T treatment, the mDOR of the subjects was 6.3 months, of which 6 patients achieved an overall survival of about 11 months or more, and 4 of them are alive and in the follow-up period. In the present invention, it has been confirmed that patients with gastric cancer / esophagogastric junction adenocarcinoma who had failed PD-1 and / or PD-L1 antibody treatment were treated with CAR-T cells, and efficacy evaluation showed complete remission (CR), and clinical observations are ongoing.

[0159] Krukenberg tumor is a metastatic malignant ovarian cancer mainly originating from the stomach and intestines. It is highly malignant and has low specificity of clinical characteristics, so it is often accompanied by systemic metastasis when discovered, and is often accompanied by ascites and diffuse peritoneal metastasis. It is insensitive to chemotherapy, has poor therapeutic effect, and has poor prognosis. In the present invention, a cell therapy product (e.g., CAR-T cells) is used for Krukenberg tumor patients who have failed PD-1 and / or PD-L1 antibody treatment, and longer progression-free survival and overall survival are shown, and the patients have obtained significant clinical benefit. That is, PR has been achieved after treatment with CAR-T cells, the progression-free survival has already exceeded 4 months, and the patient is still in a sustained remission phase.

[0160] According to currently published clinical study results, the progression-free survival period of gallbladder cancer patients who have previously received first-line standard treatment is less than about 6 months (1.8 to 5.6 months). In the present invention, a cell therapy product (exemplary, CAR-T cells) is used for gallbladder cancer patients who have failed PD-1 and / or PD-L1 antibody treatment, and longer progression-free survival and overall survival are shown, and the patients have obtained significant clinical benefit. That is, SD has been achieved with CAR-T cell therapy, and the progression-free survival period has already reached 7.5 months, and the patient is still in a sustained stable phase.

[0161] Primary liver cancer is currently the fourth most common malignant tumor in Japan and the second leading cause of tumor death. Most patients are in the late stage of the disease, and the tumor is highly deteriorated, so the therapeutic effect is not good. In response to this, the present invention uses a cell therapy product (exemplary, CAR-T cells) for liver cancer patients who have failed PD-1 and / or PD-L1 antibody treatment, and has shown longer progression-free survival and overall survival, and the patients have obtained significant clinical benefit. That is, after treatment with CAR-T cells, the disease control rate is about 54.5%, and the progression-free survival of the six patients who achieved disease control was about 4.4 months on average, and the overall survival of four of these patients exceeded one year, and three of these four cases are still alive and are in the follow-up period.

[0162] The term "cell therapy product" as used herein refers to a cell product with a medical therapeutic effect, for example, immune cell therapy products are products that use immune cells derived from the human body itself or a donor, which are injected (or implanted) into the patient's body after in vitro manipulation (including, but not limited to, separation, purification, culture, amplification, differentiation induction, activation, genetic manipulation, cell (strain) creation, cryopreservation and resuscitation), and treat diseases by inducing, enhancing or suppressing the body's immune function. These products include adoptive cell therapy (ACT) products and therapeutic vaccines. The types of immune cell therapy products mainly include, but are not limited to, Cytokine-Induced Killer (CIK), Tumor Infiltrating Lymphocyte (TIL), Chimeric Antigen Receptor T-cell (CAR-T), T Cell Receptor-engineering T-cell (TCR-T), Natural Killer (NK) cells, Dendritic cells (DC), Macrophages, etc.

[0163] The term "anti-PD-1 antibody" refers to an antibody capable of recognizing or binding to PD-1, for example, an antibody capable of recognizing or binding to human PD-1 (extracellular segment SEQ ID NO: 39). An "antibody that recognizes or binds to human PD-1" is an antibody having a binding affinity of 1.0×10 -8 It may specifically bind to human PD1 antigen with a KD value of 100 mol / L or a lower binding affinity. In one embodiment, the anti-PD-1 antibody may be Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalimab, Sintilimab, Tislelizumab, or a combination thereof.

[0164] The term "anti-PD-L1 antibody" refers to an antibody that can recognize or bind to PD-L1, such as an antibody that can recognize or bind to human PD-L1 (extracellular segment SEQ ID NO: 40). An "antibody that recognizes or binds to human PD-L1" has a binding affinity of 1.0×10 -8 It may specifically bind to the human PD-L1 antigen with a KD value of 100 mol / L or a lower binding affinity. In one embodiment, the anti-PD-L1 antibody may be Atezolizumab, Durvalumab, Avelumab, Sugemalimab (CS1001), or a combination thereof.

[0165] The term "binding" refers to selectively binding to a target. Immune effector cells that express a receptor that binds to the tumor antigen can recognize the tumor antigen.

[0166] "Specifically binds" refers to a polypeptide or a fragment thereof that recognizes and binds to a target biomolecule (e.g., a polypeptide) but does not recognize and bind to essentially other molecules in a sample.

[0167] Binding affinity may be measured by standard binding assays, for example by surface plasmon resonance technology (GE Healthcare, Uppsala, Sweden).

[0168] The "dosage" referred to herein may be a dose calculated based on body weight, a dose calculated based on body surface area (BSA), or a dose calculated on an individual basis. A dose calculated based on body weight is a dose administered to a patient calculated based on the patient's weight, e.g., mg / kg, cells / kg, etc. A dose calculated based on BSA is a dose administered to a patient calculated based on the patient's surface area, e.g., mg / m 2 , number of cells / m 2 etc. Dose calculated on an individual basis refers to the dose administered for each cycle or each time for each patient, e.g., mg / person, cell number / person.

[0169] The "number of administrations" described herein refers to the frequency with which the cell therapy product or each prior therapeutic agent is administered in each cycle. For example, fludarabine may be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day. Cyclophosphamide may be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day. Albumin-bound paclitaxel may be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day. Illustratively, the cell therapy product administered in each cycle may be administered once or may be administered 2, 3, 4, 5, 6, 7, 8, 9, 10 times, either in consecutive daily doses or at intervals of 1, 2, 3, 4, 5, 6 days.

[0170] As used herein, "response" refers to a complete response (CR) (all target lesions (lesions) disappear, and the short diameter of all pathological lymph nodes must decrease to less than 10 mm), or a partial response (PR) (the sum of the diameters of the target lesions decreases by at least 30% compared to the baseline level), or a stable disease (SD) (the degree of reduction of the target lesions does not reach PR, but the degree of increase does not reach PD level either, and is between the two, and the minimum value of the sum of the diameters may be used as the basis for the study). When evaluated as a progressive disease (PD) based on the response criteria for solid tumors v1.1 (RECIST 1.1), the minimum value of the sum of the diameters of all target lesions measured throughout the entire study must be used as the basis, and the sum of the diameters must increase by at least 20% relative to the minimum value (if the baseline measurement value is the minimum, the baseline value must be used as the basis), and the absolute value of the sum of the diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered as PD). Objective response rate (ORR) is defined as the percentage of the analyzed population who achieved a confirmed CR or PR as the best treatment response, and disease control rate (DCR) is defined as the percentage of the analyzed population who achieved a confirmed remission (CR+PR) and stable disease (SD) as the best treatment response. Progression-free survival (PFS) is defined as the time from the first dose of the patented treatment method to the first occurrence of disease progression or death from any cause. Participants who did not progress or die on the data cutoff date were excluded on the date of the last satisfactory tumor evaluation. PFS is summarized with descriptive statistics (mean, standard deviation, median, minimum and maximum) for the treated population. Except for the median, which is calculated from both observed and excluded values ​​using the Kaplan-Meier method, all other statistical indices (mean, standard deviation, minimum and maximum) are calculated only from observed values. Overall survival (OS) is measured as the time from the first dose of the patented treatment regimen to death from any cause and is analyzed in a manner similar to that described for PFS.

[0171] In some embodiments, responding includes CR or PR after treatment with the therapeutic methods of the present patents. In some embodiments, responding includes CR, PR or SD after treatment with the therapeutic methods of the present patents. In some embodiments, responding refers to subjects receiving the therapeutic methods of the present patents can obtain clear clinical benefit, and PFS and / or OS are clearly extended, for example, by 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or even longer. In some embodiments, responding refers to an ORR of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or reaching 100%. In some embodiments, a response refers to a DCR of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or reaching 100%.

[0172] The term "composition" refers to a mixture of two or more substances, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0173] A cell or cell population described herein is "positive" for a particular marker, meaning that the presence of a particular marker (generally a surface marker) on a cell can be demonstrated by detection. For example, the marker can be detected to be present on the surface by cytometry, e.g., staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining can be detected by cytometry at a level that is significantly higher than the level of staining detected when the same steps are performed using an isotype-matched control under otherwise the same conditions, and / or is substantially similar to the level of known cells that are positive for the marker, and / or is significantly higher than the level of known cells that are negative for the marker. In certain embodiments, the marker includes, but is not limited to, a tumor antigen. Exemplarily, patients with gastric / esophagogastric junction adenocarcinoma, Krukenberg tumor, liver cancer, and gallbladder cancer that are positive for the marker (tumor antigen CLDN18.2 or GPC3) receive CAR-T cell therapy after failure of PD-1 and / or PD-L1 antibody therapy.

[0174] A cell or cell population described herein being "negative" for a particular marker means that the cells do not have the particular marker or that they have undetectable surface expression, e.g., the marker is not detectable by cytometry, or surface expression is detectable, e.g., when surface expression is detected by staining with an antibody that specifically binds to the marker and detecting the antibody, the staining can be detected by cytometry at a level that is significantly lower than the level of staining detected in the same step, other conditions being the same, using an isotype-matched control, and / or significantly lower than the level of known cells that are positive for the marker, and / or substantially similar to the level of known cells that are negative for the marker. In certain embodiments, patients who are positive or negative for the marker PD-L1 in their tumor cells can both be treated with CAR-T cells after prior treatment with anti-PD-1 and / or anti-PD-L1 antibodies has failed.

[0175] The term "vector" as used herein refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, etc.

[0176] The term "cell" may refer to a cell of human or non-human animal origin.

[0177] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an individual receiving cell product therapy, e.g., a human or other animal, typically a human. Prior to enrollment in a cell therapy study, the subject may have already been treated with a PD-1 antibody and / or a PD-L1 antibody, and may be responsive or non-responsive to the PD-1 antibody or PD-L1 antibody.

[0178] The term "immune effector cell" refers to a cell that participates in an immune response and produces an immune effect, such as a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NK T) cell, a dendritic cell, a CIK cell, a macrophage, a mast cell, or a stem cell-derived immune effector cell. In some embodiments, the immune effector cell is a T cell, a NK cell, or a NK T cell. In some embodiments, the T cell may be a natural T cell and / or a T cell induced by a pluripotent stem cell. In some embodiments, the T cell may be an autologous T cell, a heterologous T cell, or an allogeneic T cell. In some embodiments, the NK cell may be an autologous NK cell or an allogeneic NK cell.

[0179] The term "artificially modified cells with immune effector cell function" refers to cells that have no immune effect, or cells that have been artificially modified or stimulated with a stimulant and then acquire the function of immune effector cells, such as 293T cells that have been artificially modified to have immune effector cell function, or stem cells that can be induced to differentiate into immune effector cells ex vivo.

[0180] In some cases, the "T cell" may be a mature T cell with immune activity that is formed by differentiation and maturation in the thymus of a pluripotent stem cell derived from bone marrow. In some cases, the "T cell" may be a cell population with a specific phenotypic characteristic or a mixed cell population with different phenotypic characteristics, for example, the "T cell" may be a cell including at least one T cell subset, i.e., stem cell-like memory T cell (Tscm cell), central memory T cell (Tcm), effector T cell (Tef, Teff), regulatory T cell (Treg), and / or effector memory T cell (Tem). In some cases, the "T cell" may be a specific subtype of T cell, for example, γδ T or αβ T cell.

[0181] T cells may be obtained from many sources, including, but not limited to, PBMCs (peripheral blood mononuclear cells), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, sites of infection, ascites, pleural effusion, spleen tissue, and tissue derived from tumors. In some cases, T cells may be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, for example, Ficoll™ separation. In one embodiment, cells are obtained from the circulating blood of an individual by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma molecules, and the cells may be placed in an appropriate buffer or medium for subsequent processing steps. Alternatively, cells may be derived from healthy donors or patients diagnosed with cancer. The T cells may be any type of T cell and at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.

[0182] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the intended function in the case of overexpression.

[0183] The term "endogenous" refers to a nucleic acid molecule, polypeptide, etc. that originates from the organism itself.

[0184] The term "chimeric receptor" refers to a fusion molecule formed by linking DNA fragments of different origins or corresponding cDNA or polypeptide fragments of proteins by genetic recombination techniques, and includes an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), chimeric T cell receptors (TCRs), and T cell-antigen couplers (TACs).

[0185] The term "chimeric antigen receptor" (CAR) comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the extracellular binding domain of the CAR comprises an scFv. The intracellular signaling domain comprises a functional domain involved in signal transduction of a stimulatory molecule and / or a costimulatory molecule, or comprises all intracellular portions of a stimulatory molecule and / or a costimulatory molecule, or all naturally occurring intracellular signaling domains, or functional fragments or derivatives thereof. In one aspect, the stimulatory molecule comprises a CD3 zeta chain that binds to the T cell receptor complex, and in one aspect, the intracellular signaling domain further comprises a functional domain involved in signal transduction of one or more costimulatory molecules, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, the polypeptide components are linked to each other. Exemplarily, the extracellular antigen binding domain of the CAR comprises a sequence as set forth in SEQ ID NO: 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.

[0186] The term "T cell receptor (TCR)" mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, and includes natural TCR receptors and modified TCR receptors. Natural TCR receptors consist of two peptide chains, α and β, each of which is further divided into a variable region (V region), a constant region (C region), a transmembrane region, a cytoplasmic region, etc., and the antigen specificity is present in the V region, and each V region (Vα, Vβ) has three hypervariable regions CDR1, CDR2, and CDR3. In certain embodiments, the modified TCR extracellular antigen-binding domain comprises an antibody that recognizes a tumor antigen. Exemplarily, the TCR has a sequence as shown in SEQ ID NO: 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.

[0187] The term "TCR fusion protein" or "TFP" includes recombinant proteins of various proteins derived from TCR, and generally includes an antigen-binding domain that is capable of i) binding to a surface antigen on a target cell and ii) interacting with other polypeptide components of the intact TCR complex when localized in a T cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell. In some embodiments, the TFP T cell is a NK T cell. In some embodiments, the TFP T cell is a γδ T or an αβ T cell. In certain embodiments, the antigen-binding domain of the TFP includes an antibody that recognizes a tumor antigen. Exemplarily, the TFP comprises a sequence as set forth in SEQ ID NO: 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79.

[0188] The term "T cell antigen coupler (TAC)" comprises three functional domains: 1. an antigen-binding domain, which may comprise a single chain antibody, a designed ankyrin repeat protein (DARPin) or other targeting group; 2. an extracellular domain, which is a single chain antibody that binds to CD3, bringing the TAC receptor into close proximity with the TCR receptor; and 3. a transmembrane domain and an intracellular domain of the CD4 co-receptor, which is linked to the protein kinase LCK and catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the first step in T cell activation. In certain embodiments, the TAC antigen-binding domain comprises an antibody that recognizes a tumor antigen. Exemplarily, the TAC has a sequence as shown in 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.

[0189] The term "antigen-binding domain" refers to the immunologically active portion of an immunoglobulin molecule (which may be used interchangeably with "antibody") or immune molecule, i.e., a molecule that contains an antigen-binding site that specifically binds to an antigen ("immunoreactive"), and may include antibodies such as fully human antibodies, chimeric antibodies, humanized antibodies, murine antibodies, or may be a ligand that recognizes an antigen.

[0190] The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. As used herein, the term "antibody" refers to an antigen-binding protein of the immune system. "Antibody" includes intact full-length antibodies with an antigen-binding region and any fragment thereof in which the "antigen-binding portion" or "antigen-binding region" thereof is retained, or a single chain thereof, e.g., a single-chain variable fragment (scFv). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The term "variable region or variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.

[0191] Antibody fragments include (i) Fab fragments (including Fab' and Fab'-SH) consisting of the VL, VH, CL, and CH1 domains, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody, (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546), (v) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments, and (vi) the antigen-binding site of a single chain Fv molecule (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883), (vii) bispecific single chain Fv dimers (PCT / US92 / 09965), (viii) "dibodies" or "triabodies" which are multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448), and (ix) scFvs fused to genes of the same or different antibodies (Coloma & Morrison, 1997, Nature Biotechnology 15,159-163).

[0192] In certain embodiments, the antigen-binding domain may comprise an scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain fragment, or a natural ligand that binds to the antigen, and any derivative thereof. In some aspects, the extracellular antigen-binding domain (e.g., scFv) may comprise light chain CDRs and / or heavy chain CDRs specific for the antigen.

[0193] In a preferred embodiment, the antibody or fragment thereof comprised in the antigen-binding domain may comprise the following CDR sequences: HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3, LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, LCDR3 as set forth in SEQ ID NO: 6, or HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18, LCDR1 as set forth in SEQ ID NO: 19, LCDR2 as set forth in SEQ ID NO: 20, LCDR3 as set forth in SEQ ID NO: 21, or HCDR1 as set forth in SEQ ID NO: 27, HCDR2 as set forth in SEQ ID NO: 28, HCDR3 as set forth in SEQ ID NO: 29, LCDR1 as set forth in SEQ ID NO: 30, LCDR2 as set forth in SEQ ID NO: 31, LCDR3 as set forth in SEQ ID NO: 32.

[0194] In a preferred embodiment, the antibody or fragment thereof comprised in the antigen-binding domain may comprise the following heavy and light chain variable region sequences: a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:9, or a heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region set forth in SEQ ID NO:23, or a heavy chain variable region set forth in SEQ ID NO:33 and a light chain variable region set forth in SEQ ID NO:34.

[0195] In certain embodiments, the antibody or fragment thereof comprised in the antigen binding domain may comprise an scFv sequence as set forth in SEQ ID NO:58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.

[0196] In some cases, the extracellular region of the CAR comprises an antigen-binding domain that recognizes a tumor antigen and a linker fragment (also called a hinge or spacer region). The linker fragment may be considered as part of the CAR to provide flexibility to the extracellular antigen-binding domain. The linker fragment may be of any length. Exemplarily, in one embodiment, the CAR comprises one hinge domain, illustratively the CD8α hinge shown in SEQ ID NO:41.

[0197] The term "transmembrane domain" can anchor an exogenous receptor to the plasma membrane of a cell. For example, the transmembrane domain of CD28, CD8, comprising the sequence set forth in SEQ ID NO: 42 or 43, can be used. The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell, acting as an effector by generating or responding to second messengers, and is used to regulate the activity of a cell by a determined signaling pathway. The intracellular signaling domain may include the entire intracellular portion of a molecule, or all naturally occurring intracellular signaling domains, or functional fragments or derivatives thereof. Exemplarily, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 44, 45, or 46.

[0198] Functional domains involved in signal transduction of stimulatory molecules are also called "primary signal domains" and stimulatorily regulate the initial activation of CAR or TCR complexes. In one embodiment, the primary signal domain is induced, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. Primary signal domains that act in a stimulatory manner may include immunoreceptor tyrosine-based activation motifs or signal transduction motifs of ITAMs. Examples of primary signal domains containing ITAMs that are particularly useful in the present application include, but are not limited to, sequences derived from CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), and CD66d.

[0199] The term "costimulatory molecule" refers to a cell stimulatory signal molecule, such as one that binds to TCR / CD3 and in combination causes a signal for T cell proliferation and / or up-regulation or down-regulation of key molecules, and has the sequence shown in SEQ ID NO: 44 or 45. It is the cognate binding partner in T cells, which specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, including but not limited to cell proliferation. A costimulatory molecule is a non-antigen receptor cell surface molecule or its ligand that is required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137).

[0200] In some cases, the intracellular signaling domain may be designed to include several potential stimulatory molecules and / or costimulatory signal domains. Illustratively, a first generation CAR includes a single stimulatory domain, such as CD3 zeta chain, a second generation CAR includes one costimulatory domain, such as CD3 zeta chain and CD28, or CD3 zeta chain and 4-1BB, and a third generation CAR includes two costimulatory domains (third generation CAR), such as CD3 zeta chain and CD28, OX40, or CD3 zeta chain and CD28, 4-1BB.

[0201] In certain embodiments, the CAR comprises an optional leader sequence. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during the process of cellular processing and localization of the CAR to the cell membrane. In one embodiment, the leader sequence comprises the sequence shown in SEQ ID NO:53.

[0202] In a particular embodiment, the antigen-binding domain targeting the GPC3-CAR of the present invention comprises a sequence as set forth in SEQ ID NO: 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79, the transmembrane domain comprises a sequence as set forth in SEQ ID NO: 42 or 43, and the intracellular signaling domain comprises a sequence as set forth in SEQ ID NO: 44 and 46, or SEQ ID NO: 45 and 46, or SEQ ID NO: 45, 44 and 46. Illustratively, administration of the GPC3-CAR-T cells to liver cancer patients who have failed PD1 and / or PD-L1 antibody treatment has shown longer progression-free survival and overall survival, and the patients have obtained significant clinical benefit. In an embodiment described in the present invention, administration of the GPC3-CAR-T cells and the transcription factor RUX3 polypeptide to liver cancer patients who have failed PD1 and / or PD-L1 antibody treatment has shown significant clinical benefit, with some patients being evaluated as PR after treatment. In a particular embodiment, the GPC3-CAR-T expresses RUNX3 polypeptide.

[0203] The term "RUNX3 (Runt-related transcription factor 3)", also known as osteogenesis-related transcription factor 3, is a member of the runt domain family of transcription factors and has the amino acid sequence GenBank Accession No. NP_004341.1. It plays an important role in cell growth, development, apoptosis, signal transduction and its biological effects.

[0204] The terms "activation" and "activation" may be used interchangeably and may refer to the process by which cells change from a quiescent state to an active state. The process may include a response to an antigen, migration, and / or a phenotypic or genetic change to a functionally active state. For example, the term "activation" may refer to the process by which T cells are gradually activated. The activation process is controlled by both primary and costimulatory signals. "Activation of T cells" refers to the state in which T cells are stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. The use of CD3 / CD28 magnetic beads, ex vivo antigen stimulation, or in vivo antigen stimulation all affect the degree and duration of T cell activation. In one embodiment, the engineered T cells are activated after co-incubation with tumor cells containing a specific target antigen or infection with a virus.

[0205] The term "treatment" refers to a procedure that seeks to change the course of a disease, alleviating or reducing the disease or symptoms associated therewith, in whole or in part. The effect of said treatment includes, but is not limited to, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, mitigation of the rate of progression of the disease, improvement or alleviation of the disease state, alleviation or improvement of the prognosis. As used herein, "delaying tumor progression" refers to delaying, hindering, alleviating, reducing, stabilizing, inhibiting and / or postponing the progression of said tumor. The delay may comprise different lengths of time, which is determined by the medical history and / or the individual being treated. It should be understood by those skilled in the art that a sufficient or significant delay may also cover prevention (in the case of an individual in which the tumor has not progressed). For example, late cancer (e.g., progression of metastasis) may be delayed.

[0206] In some embodiments, the cell therapy products provided are used to slow or ameliorate the progression of tumors.

[0207] As used herein, "inhibition" refers to a decrease in function or activity when compared to the same condition in other circumstances or when compared to another condition.

[0208] An "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat a disease (cancer) in an individual. The effective dose for therapeutic or prophylactic use will depend on the stage and severity of the disease being treated, the age, weight, and general health of the recipient, and the judgment of the prescribing physician. The amount of dose will also depend on the active agent selected, the method of administration, the duration and frequency of administration, the existence, nature and extent of any adverse side effects associated with the administration of a particular active agent, and the physiological effect desired. At the discretion of the prescribing physician or one of skill in the art, it may be necessary to administer the cell therapy products described herein in one or more cycles, or multiple times.

[0209] The term "tumor antigen" refers to an antigen that is newly emerged or overexpressed during the development or progression of a hyperproliferative disease. In some embodiments, the hyperproliferative condition of the present invention refers to cancer (including tumors). The tumor antigen according to the present invention may be a solid tumor antigen or a hematological tumor antigen.

[0210] The term "cycle" refers to the period from the baseline period before each cell therapy treatment (i.e., the evaluation phase) to the baseline period before the next treatment, e.g., from administration of a prior therapeutic drug to before the start of the next cycle of prior therapeutic drug treatment.

[0211] The term "pretreatment" refers to administering other drugs or treatments to a patient before administering a product (e.g., a cell therapy product) to make the patient's physical condition more suitable for the product treatment. For example, a chemical drug, a biological drug, a radiation therapy, or a combination thereof is administered before administering a cell therapy product (e.g., CAR-T cells) to perform pretreatment. Preferably, a chemical drug is administered first to perform pretreatment, and more preferably, the chemical drug is a chemotherapeutic drug.

[0212] In some embodiments, any pretreatment is required prior to administration of the cell therapy product in each cycle.

[0213] In some embodiments, after pretreatment, the patient has about a 50%, about 55%, about 60%, about 65%, or about 70% decrease in lymphocytes compared to before pretreatment.

[0214] The term "chemical agent" or "chemical drug" refers to an anti-cancer agent produced by the method of chemical synthesis, which may be a conventional chemotherapeutic agent, such as an alkylating agent, antimetabolite, anti-cancer antibiotic, etc., or a targeted drug, such as afatinib, alectinib, etc.

[0215] The term "biologic drug" or "biologic drug" refers to a drug produced by molecular biology or genetic engineering means, such as an antibody drug or an ADC.

[0216] The term "chemotherapeutic agent" refers to a drug that treats tumors. Chemotherapeutic agents can kill tumor cells or inhibit tumor cell growth, migration, invasion, etc. These drugs act at different stages of tumor cell growth and proliferation, and can inhibit or kill tumor cells. Treatment with chemotherapy agents is currently one of the main means of treating tumors.

[0217] In some embodiments, the chemotherapeutic agent described herein refers to a drug used in chemotherapy, and refers to a chemical drug that has a preventive or therapeutic effect on malignant tumors. Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, plant-based anticancer agents, hormones, immunosuppressants, etc. For example, diterpene alkaloid compounds (e.g., taxanes), cyclophosphamide, fludarabine, cyclosporine, rapamycin, melphalan, bendamustine, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, rituximab, vinblastine, and / or vincristine, etc.In some embodiments, the antimetabolite is, for example, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine sodium octadecyl phosphate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emiteflu, tiazofur in), nolatrexed, pemetrexed, nelarabine, 2'-deoxy-2'-methylenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienyl]glycylamino]-L-glycerol-BL-mannose-hep topyranosyl]adenine, aplidine, ecteinascidin, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa- Examples of the antimetabolites include, but are not limited to, 1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetate, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosyl cytosine, and 3-aminopyridine-2-aldehyde aminothiourea. In some embodiments, the alkylating agents include, but are not limited to, dacarbazine, melphalan, cyclophosphamide, temozolomide, chlorambucil, busulfan, nitrogen mustard, and nitrosoureas.

[0218] Uses or Methods of Cell Therapy Products: The present invention provides for the use of cell therapy products or methods for the treatment of cancers (used interchangeably with "tumor") that have failed prior treatment with anti-PD-1 and / or anti-PD-L1 antibodies.

[0219] In some embodiments, at least 40% of patients who have failed prior treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody respond to the cell therapy product.

[0220] In some embodiments, at least 50% of patients who have failed prior treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody respond to the cell therapy product.

[0221] In some embodiments, after administration of the cell therapy product, the patient has an ORR of greater than 40%, preferably greater than 50%.

[0222] As used herein, "dose" refers to the amount of pharmaceutical agent administered, e.g., the amount of a cell therapy product administered within a cycle, or the amount of a cell therapy product administered each time within a cycle, or the amount of a prior therapeutic agent administered each time within a cycle.

[0223] In some embodiments, the patient is treated with at least one cycle of a cell therapy product, and in certain embodiments, the dose of each cycle may be the same or different.

[0224] In some embodiments, a cycle of doses is administered to a subject one or more times, and the fractional doses of each administration may be the same or different, for example, a cycle of a cell therapy product is administered to a subject once, or is administered to a subject two, three, four, five, or more times.

[0225] In some embodiments, when a cycle of doses is administered to a candidate multiple times, the dose of the cell therapy product or the previous therapeutic agent administered each time is determined by the physician based on the candidate's specific circumstances. The candidate's specific circumstances may be the candidate's overall health status, the severity of the disease, the response to the previous dose of the same cycle, the response to the previous cycle, the candidate's drug concomitant status, the degree or possibility of adverse reactions, complications, the metastatic status of the cancer, and any other factors that the physician believes will affect the dose of the cell therapy or the previous therapeutic agent that the candidate receives. In some embodiments, the dose of each administration tends to increase gradually. In some embodiments, the dose of each administration tends to decrease gradually. In some embodiments, the dose of each administration tends to increase gradually and then decrease gradually. In some embodiments, the dose of each administration tends to decrease gradually and then increase gradually.

[0226] Administering multiple cycles of a cell therapy product refers to administering a fixed total amount of cell therapy product during multiple time periods. In some embodiments, the time periods are coincident. In some embodiments, the time periods are not coincident. In some embodiments, at least two cycles of the cell therapy product are administered, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of the cell therapy product are administered.

[0227] In some embodiments, the dose of cells administered per cycle and the number of administrations, and the spacing of different cycles, are evaluated with the goal of improving one or more outcomes, for example, an outcome may be reducing the severity or likelihood of a side effect in a subject, or an outcome may be improving the efficacy of treatment.

[0228] In some embodiments, the dose of cells in the cell therapy product administered per cycle is about 2×10 9 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 11Preferably, the dose of cells in the cell therapy product administered per cycle is about 2×10 8 Do not exceed 1 x 10 cells / kg of patient body weight, or the cell dose in the cell therapy product is approximately 1 x 10 10 More preferably, the dose of cells in the cell therapy product administered per cycle is about 2×10 7 cells / kg of patient body weight, or the dose of cells in the cell therapy product is approximately 5 x 10 9 cells / person, or approximately 2 x 10 9 cells / person, or approximately 1 x 10 9 cells / person, or approximately 5 x 10 8 Not to exceed 10 cells / person.

[0229] In some embodiments, the dose of cells in the cell therapy product administered per cycle is about 1×10 5 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of examinee's body weight, or approximately 1 x 10 6 cells / kg of examinee's body weight ~ approx. 2 x 10 7 cells / kg of examinee's body weight.

[0230] In some embodiments, the dose of cells in the cell therapy product administered per cycle is about 1×10 7 ~ approx. 1 x 10 cells 9 cells, approximately 1 x 10 7 ~ approx. 2 x 10 cells 9 cells, approximately 1 x 10 7 ~ approx. 5 x 10 cells 9 cells, or approximately 1 x 10 7 ~ approx. 1 x 10 cells 10 cells, preferably about 1×10 8 ~ approx. 1 x 10 cells 9 cells, approximately 1 x 10 8 ~ approx. 2 x 10 cells 9 cells, approximately 1 x 10 8 ~ approx. 5 x 10 cells 9 cells, more preferably about 1×10 8~ approx. 5 x 10 cells 8 cells, or approximately 2.5 x 10 8 ~ approx. 5 x 10 cells 8 cells, approximately 3.75 x 10 8 ~ approx. 5 x 10 cells 8 Each cell is a single cell.

[0231] In some embodiments, prior to administration of the cell therapy product in each cycle, the patient is pretreated, the pretreatment including administering to the patient a chemotherapy agent and / or radiation therapy, which in some embodiments may be systemic or localized radiation therapy.

[0232] In some embodiments, the pretreatment chemotherapeutic agent may be any one or at least two of cyclophosphamide, fludarabine, a tubulin inhibitor, and a pyrimidine antitumor agent.

[0233] The tubulin inhibitors described herein include tubulin polymerization promoters and tubulin polymerization inhibitors, including, but not limited to, taxanes, epothilones, discodermolides, laulimalides, and the like.

[0234] The term "taxanes" refers to drugs that contain a taxane compound as a major component, the taxane compound comprising a bridged methanobenzocyclodecene core structure similar to taxane. In some embodiments, the bridged methanobenzocyclodecene core structure of the taxane compound contains an unsaturated bond. In some embodiments, the bridged methanobenzocyclodecene core structure of the taxane compound does not contain an unsaturated bond. In some embodiments, a carbon atom in the bridged methanobenzocyclodecene core structure of the taxane is replaced by a heteroatom selected from N, O, S, and P. In some embodiments, the taxane compound is administered by injection.

[0235] In some embodiments, the prior chemotherapy agents may be cyclophosphamide and fludarabine, or cyclophosphamide, fludarabine and a tubulin inhibitor.

[0236] In some embodiments, the tubulin inhibitor is a taxane compound, preferably the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably the taxane compound is albumin-bound paclitaxel.

[0237] In some embodiments, the pyrimidine antitumor agent is selected from 5-fluorouracil, gimeracil, oteracil potassium, tegadiful, carmofur, doxifluridine, and capecitabine.

[0238] In some embodiments, the dosage of fludarabine is about 20-60 mg / day, or about 30-55 mg / day, or about 30-50 mg / day.

[0239] In some embodiments, the dosage of fludarabine is about 10-50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or about 25 mg / m 2 / day.

[0240] In some embodiments, the dosage of cyclophosphamide is about 200-700 mg / day, or about 300-600 mg / day, or about 300-560 mg / day, or about 300-550 mg / day, or about 300-500 mg / day.

[0241] In some embodiments, the dose of cyclophosphamide is about 200-400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day.

[0242] In some embodiments, the dosage of the taxane compound is about 300 mg / day or less, or about 200 mg / day or less, preferably, the dosage of the taxane compound is about 90-200 mg / day, and more preferably, the dosage of the taxane compound is about 90-120 mg / day.

[0243] In some embodiments, consecutive use of each chemotherapy agent does not exceed 4 days.

[0244] In some embodiments, cyclophosphamide is administered 2-3 times. In some embodiments, fludarabine is administered 1-2 times. In some embodiments, taxane compound is administered once.

[0245] In some embodiments, the methods or uses provided herein are derived from the applicant's observed responses and treatment outcomes of subjects to administered cell therapy products. When CAR-T cells targeting CLDN18A2 are administered at different doses or different cycles to subjects with CLDN18A2-positive cancer who have previously failed treatment with anti-PD-1 and / or anti-PD-L1 antibodies, the CAR-T cells can still show good treatment outcomes. When CAR-T cells targeting GPC3 are administered at different doses or different cycles to subjects with GPC3-positive cancer who have previously failed treatment with anti-PD-1 and / or anti-PD-L1 antibodies, the CAR-T cells can still show good treatment outcomes. When GPC3-CAR-T cells expressing RUNX3 are administered at different doses or different cycles to subjects with GPC3-positive cancer who have previously failed treatment with anti-PD-1 and / or anti-PD-L1 antibodies, the CAR-T cells can still show good treatment outcomes. For example, these treatment outcomes include patient survival, remission, or stable disease.

[0246] In some embodiments, the uses or methods described herein further comprise monitoring the extent or risk of an adverse reaction to the cell therapy product to the recipient and determining the administration dose and interval of subsequent multiple administrations or subsequent cycles based on the extent or risk of said adverse reaction, which in some embodiments includes, but is not limited to, CRS, neurotoxicity, macrophage activation syndrome, tumor lysis syndrome, and the like.

[0247] In some embodiments, subsequent cycles of treatment are administered when the risk of an adverse reaction or its symptoms or biochemical indicators (e.g., CRS or neurotoxicity, macrophage activation syndrome or tumor lysis syndrome) is equal to or less than an acceptable level, generally following administration of a cell therapy product. In some embodiments, the adverse reaction or its symptoms or biochemical indicators include one or more of fever, hypotension, anoxia, neuropathy, inflammatory cytokines, and serum levels of C-reactive protein (CRP). In some embodiments, an acceptable level of risk of the adverse reaction or its symptoms or biochemical indicators refers to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the peak level following administration of a previous cycle of treatment.

[0248] In some embodiments, a subsequent cycle of treatment is administered if, after administration of the previous cycle of cell therapy product, the serum level of a factor indicative of cytokine release syndrome (CRS) in the subject is not more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times the serum level in the subject before administration of the previous cycle.

[0249] In some embodiments, the timing of administration of the later cycle is selected to avoid the possibility that the host's immune response already generated by administration of the previous cycle will induce a decrease in the effectiveness of the later cycle. In some aspects, the later cycle is administered before the appearance of a host's immune response, e.g., an adaptive or specific immune response to the administered cells and / or the chimeric antigen receptor expressed thereby, e.g., a humoral or cell-mediated immune response. In some embodiments, the later cycle is administered before such a response can be detected, e.g., by one or more specialized detection methods. In general, one or more later cycles are administered when the host's adaptive immune response to the cells has not been detected, has not yet been established, and / or has not yet reached a certain level or degree or stage.

[0250] In some embodiments, after a prior cycle of treatment, the tumor burden becomes stable or decreases, and before the host's adaptive immune response (i.e., the body's rejection reaction to the CAR-T cells) occurs, one or more subsequent cycles of treatment are administered. In such conditions, the subsequent cycles can safely and effectively provide immune surveillance to eliminate or prevent the growth or metastasis of remaining tumor cells. Thus, in some embodiments, the subsequent cycles are consolidation doses.

[0251] In some embodiments, the subsequent cycle of treatment is administered at least 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days after administration of the previous cycle of cell therapy product.

[0252] The term "tumor burden" includes the volumetric size or degree of differentiation of a tumor, or the type, stage, and / or appearance and disappearance of complications common to intermediate or late stage cancer, such as cancerous pleural or ascites effusion, and / or the appearance or change in expression levels of tumor markers, and / or the likelihood or incidence of adverse effects in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or host immune response to the administered cells and / or chimeric antigen receptor. In some embodiments, tumor size is measured by measures provided by PET (positron emission tomography) and CT (computed tomography).

[0253] The tumor marker is also called a tumor marker, and refers to a substance that is characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or produced by the host in response to tumor stimulation, which reflects the occurrence and progression of tumors and can monitor the response of tumors to treatment. Tumor markers are present in the tissues, body fluids, and excretions of tumor patients and can be detected by immunological, biological, and chemical methods, and examples thereof include alpha fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma-associated antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

[0254] The uses or methods provided by the present invention include administering at least one cycle of immune effector cells expressing, for example, a CAR, a TCR, a TFP, or a TAC, or a combination thereof, as a cell therapy product to a cancer patient who has failed prior treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

[0255] As used herein, a "tumor antigen" includes thyroid stimulating hormone receptor (TSHR), CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD22, CD30, CD70, CD123, CD138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, KIT (CD117), interleukin 13 receptor, and the like. Interleukin-13Rα, interleukin-11 receptor α (IL-11Rα), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, serine protease 21 (PRSS21), vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), cell surface mucin 1 (MUC1) and MUC6, epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), neural cell adhesion molecule (NCAM), carbonic anhydrase IX (CAIX), LMP2, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer, TGS5, high molecular weight melanoma-associated antigen (HMWMAA), orthoacetylglucosamine (OAcGD2) ganglioside, folate receptor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related protein (TEM7R), Claudin 6 and Claudin18.2 and Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, integrin αvβ6, B cell maturation antigen (BCMA), CA9, kappa light chain, CSPG4, EGP2 and EGP40, FAP, FAR, FBP, fetal AchR, HLA-A1 and HLA-A2, MAGEA1 and MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, survivin, TAG72, TEM1, fibronectin, cytotactin, oncofetal variants of intratumoral necrotic areas,G protein-coupled receptor class C group 5 member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific protein 1 (PLAC1), hexose moiety of globo H glycoceramide (Globo H), breast cancer-associated antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), β3-adrenergic receptor (ADRB3), pannexin 3 (pannexin 3, PANX3), G protein-coupled receptor 20 (GPR20), locus K9 of lymphocyte antigen 6 complex (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), ETS translocation variant gene 6 (ETV6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiogenin-binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, p53 mutant, human telomerase reverse transcriptase (hTERT), sarcoma breakpoint region, inhibitor of apoptosis in melanoma (ML-IAP), ERG (transmembrane serine protease 2 (TMPRSS2) ETS fusion gene), N-acetylglucosamine esterase (EGFR) fusion gene, N-acetylglucosamine esterase (N-acetylglucosamine esterase ... Glucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, V-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog gene family member C (RhoC), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS), squamous cell carcinoma-associated antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OYTES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma, breakpoint X2 (SSX2), CD79a, CD79b, CD72,These include, but are not limited to, leukocyte immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR), leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like protein 5 (FCRL5), immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is EGFR, EGFRvIII, glypican 3, claudin 18.2, or BCMA.

[0256] In some embodiments, the tumor antigen may be at least one of CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2, GPC3, IL13RA2.

[0257] In some embodiments, the tumor antigen may be at least one of CD19, BCMA, Claudin18.2, GPC3.

[0258] In some embodiments, the tumor or cancer is selected from the group consisting of colon cancer, gallbladder cancer, Krukenberg tumor, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's lymphoma, The cancer may be a non-Hodgkin's lymphoma, a neuroendocrine tumor, a thyroid cancer, a parathyroid cancer, an adrenal cancer, a soft tissue sarcoma, a urethral cancer, a penile cancer, a childhood solid tumor, a bladder cancer, a kidney or ureter cancer, a renal pelvis cancer, a central nervous system (CNS) tumor, a primary CNS lymphoma, a tumor angiogenesis, a spinal tumor, a brain stem glioma, a pituitary adenoma, a Kaposi's sarcoma, an epidermoid carcinoma, a squamous cell carcinoma, a T-cell lymphoma, an environmentally induced cancer, a combination of the cancers, and a metastatic lesion of the cancer. In some embodiments, the gastric cancer comprises an esophagogastric junction adenocarcinoma. In some embodiments, the liver cancer comprises a hepatocellular carcinoma.

[0259] CLD18 and CLD18-positive tumors: Claudin 18 (CLD18) molecule (Genbank accession numbers: splice variant 1 (Claudin 18.1, CLD18A1): NP_057453, NM016369, and splice variant 2 (Claudin 18.2, CLD18A2): NM_001002026, NP_001002026)) is an integral transmembrane protein with a molecular weight of about 27.9 / 27.72 kD. Claudins are integral membrane proteins located in the tight junctions of epithelial and endothelial cells. In a particular embodiment, CLD18A2 comprises the sequence shown in SEQ ID NO: 56. CLD18A2 is highly expressed in several types of cancer, including gastric / esophagogastric junction adenocarcinoma, esophageal cancer, gallbladder cancer, pancreatic cancer, lung cancer, and Krukenberg tumors. Expression is primarily in the adenocarcinoma subtype for these indications.

[0260] GPC3 and GPC3-positive tumors: As used herein, "GPC3" or "Glypican 3" is a member of the Glypican family that plays an important role in controlling cell growth and differentiation. In certain embodiments, GPC3 has a sequence as set forth in SEQ ID NO: 68. Abnormal expression of GPC3 is closely related to the development and progression of multiple tumors, and is abnormally expressed in, for example, liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, etc.

[0261] Methods of administering cell therapy products are known. In some embodiments, the cell therapy product (e.g., CAR-T cells) may be administered to a patient in an autologous reinfusion manner. Thus, in some aspects, the cell therapy product is derived from a subject in need of the cell therapy product and administered to the same subject after ex vivo genetic modification. In some embodiments, the cell therapy product (e.g., CAR-T cells) may be administered to a patient in an allogeneic reinfusion manner. Thus, in some other aspects, the cell therapy product is derived from a healthy donor, the donor being different from the subject in whom the cells are reinfused. In these aspects, the cell therapy product is administered to a genetically histocompatible subject, e.g., the donor and the subject are genetically the same or similar. In some embodiments, the donor and the subject have the same HLA class or supertype.

[0262] The cells can be administered in any suitable manner, for example, by injection (e.g., intravenous or subcutaneous injection, intraperitoneal injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, retro-interval injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, episcleral injection, retrobulbar injection, periocular injection or periocular delivery). In some embodiments, they may be administered parenterally, intrapulmonary and intranasally, intralesionally when localized treatment is desired. Extraperitoneal injection includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0263] When preventing or treating a disease, the appropriate dosage may depend on the type of disease being treated, the type of chimeric antigen receptor or cells, the severity and course of the disease, whether the cells are being administered for prophylactic or therapeutic purposes, previous treatments, the subject's medical history and response to the cells, and the judgment of the attending physician. In some embodiments, the compositions and cells are suitable for administration to the subject at one time or as a series of treatments.

[0264] Pretreatment prior to administration of a cell therapy product can improve the efficacy of immune effector cell therapy. Pretreatment is administered prior to infusion of immune effector cells, with day 0 being the day of the first infusion of immune effector cells (e.g., CAR-T cells) in each cycle of administration. In some embodiments, pretreatment is administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to administration of adoptive cells or immune effector cells. In some embodiments, pretreatment is administered 2-8 days prior to administration of a cell therapy product, and preferably 3-6 days prior to administration of a cell therapy product. As described above, day 0 is the day that the patient is administered CAR-T cell therapy in each cycle. In some embodiments, pretreatment may be administered at any time prior to administration of CAR-T cell therapy.

[0265] In some embodiments, the efficacy of the cell therapy product can be maximized by adjusting the duration or frequency of administration of the chemotherapeutic agent or treatment administered in the pretreatment.

[0266] In some embodiments, the cyclophosphamide is administered 2-3 times, or the fludarabine is administered 1-2 times, or the albumin-bound paclitaxel is administered once.

[0267] In some embodiments, consecutive use of each chemotherapy agent does not exceed 4 days.

[0268] In some embodiments, the dosage of fludarabine is about 20-60 mg / day, or about 30-60 mg / day, or about 30-55 mg / day, or about 30-50 mg / day.

[0269] In some embodiments, the dosage of fludarabine is about 10-50 mg / m 2 / day, or about 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, or about 25 mg / m 2 / day.

[0270] In some embodiments, the dosage of cyclophosphamide is about 200-700 mg / day, or about 300-700 mg / day, or about 300-600 mg / day, or about 300-560 mg / day, or about 300-550 mg / day, or about 300-500 mg / day.

[0271] In some embodiments, the dose of cyclophosphamide is about 200-400 mg / m 2 / day, or about 200-300 mg / m 2 / day, or approximately 250 mg / m 2 / day.

[0272] In some embodiments, the dosage of the taxane compound is about 300 mg / day or less, or about 200 mg / day or less, preferably, the dosage of the taxane compound is about 90-200 mg / day, and more preferably, the dosage of the taxane compound is about 90-120 mg / day.

[0273] The day on which the patient receives CAR-T cell therapy in each cycle is designated as day 0. In some embodiments, the patient is administered fludarabine on day -6 and day -5. In some embodiments, the patient is administered fludarabine on day -5 and day -4. In some embodiments, the patient is administered fludarabine on day -4 and day -3. In some embodiments, the patient is administered fludarabine on day -6, day -5, day -4 and day -3. In some embodiments, the patient is administered fludarabine on day -7, day -6, day -5 and day -4. In some embodiments, the patient is administered cyclophosphamide on day -6, day -5, day -4 and day -3. In some embodiments, the patient is administered cyclophosphamide on day -6, day -5 and day -4. In some embodiments, the patient is administered cyclophosphamide on day -5, day -4 and day -2. In some embodiments, the patient is administered cyclophosphamide on day -5, day -4, and day -3. In some embodiments, the patient is administered cyclophosphamide on day -4, day -3, and day -2. In some embodiments, the patient is administered cyclophosphamide on day -4 and day -3. In some embodiments, the patient is administered cyclophosphamide on day -6 and day -5. In some embodiments, the patient is administered cyclophosphamide on day -5 and day -4. In some embodiments, the patient is administered a taxane compound (e.g., albumin-bound paclitaxel) on day -2, day -3, day -4, day -5, or day -6.

[0274] Fludarabine, cyclophosphamide and albumin-bound paclitaxel can be administered on the same or different days. When fludarabine, cyclophosphamide and albumin-bound paclitaxel are administered on the same day, cyclophosphamide and / or albumin-bound paclitaxel can be administered before or after fludarabine, or fludarabine and / or albumin-bound paclitaxel can be administered before or after cyclophosphamide, or cyclophosphamide and / or fludarabine can be administered before or after albumin-bound paclitaxel.

[0275] In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel can be administered simultaneously or sequentially. In some embodiments, the patient is administered cyclophosphamide before administering fludarabine. In some embodiments, the patient is administered cyclophosphamide after administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel before administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel after administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel before administering cyclophosphamide. In some embodiments, the patient is administered albumin-bound paclitaxel after administering cyclophosphamide.

[0276] Fludarabine, cyclophosphamide and albumin-bound paclitaxel may be administered by any route, including intravenous (IV).

[0277] The methods described herein may include various other treatments. For example, after administration of cyclophosphamide and fludarabine, adverse events may occur in the patient. The scope of the present invention includes reducing some of these adverse events by administering a composition to the patient. In some embodiments, the method includes administering saline to the patient. Saline may be administered to the patient before or after administration of cyclophosphamide and / or fludarabine, or before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, saline is administered to the patient before administration of cyclophosphamide and / or fludarabine and after administration of cyclophosphamide and / or fludarabine on each infusion day. The patient may also be administered adjuvants and excipients, such as mesna (sodium 2-mercaptoethanesulfonate). Additionally, the patient may be administered exogenous cytokines.

[0278] After administering the cell therapy product to the subject, the biological activity of the engineered immune effector cell population can be measured by any of a variety of known methods. Parameters for evaluation include specific binding of the cells to antigen in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or cytometry). In some embodiments, the ability of the engineered immune effector cells to destroy target cells can be detected by any suitable method known in the art, for example, cytotoxicity assays as described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009); Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In some embodiments, the biological activity of the immune effector cells can be measured by measuring the expression and / or secretion of certain cytokines, for example, CD107a, IFNγ, IL-2, TNF. In some aspects, the biological activity is obtained by evaluating clinical outcomes, for example, tumor burden or reduction in burden. In some aspects, reduction in tumor markers is evaluated. In some embodiments, the toxicity outcome, persistence and / or proliferation of cells, and / or the presence or absence of a host immune response is assessed.

[0279] As used herein, a "previous cycle" refers to an administration cycle that precedes the administration of a later cycle, as opposed to a "later cycle". For example, when the administration of the first cycle is completed, followed by the administration of the second cycle and the third cycle, between the second cycle and the first cycle, the first cycle is the previous cycle and the second cycle is the later cycle, and between the third cycle and the second cycle, the second cycle is the previous cycle and the third cycle is the later cycle.

[0280] In certain embodiments, the dose of a cell therapy product refers to the number of cells contained in the cell therapy product.

[0281] In some embodiments, the amount or magnitude of the subsequent cycle is sufficient to reduce tumor burden or indicators thereof, and / or one or more symptoms of a disease or condition. In some embodiments, the dose can effectively improve the survival of the subject, e.g., improve the survival of the subject, e.g., induce survival, recurrence-free survival, or event-free survival of the subject to last at least 1 month or at least 1 year, 2 years, 3 years, 4 years, or 5 years.

[0282] In some embodiments, the reduction in tumor burden, including tumor size, tumor volume and / or tumor mass, after a subsequent cycle compared to the time point before administration of the previous or subsequent cycle is equal to or about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.

[0283] In other embodiments, the number of immune effector cells administered in a subsequent cycle is less than the number of immune effector cells administered in a previous cycle.

[0284] In some embodiments, the decision to administer a subsequent cycle of treatment is based on one or more criteria, e.g., on the subject's response to a previous cycle of treatment; in the case of chemotherapy, the subject's tumor burden, e.g., tumor volume, size or degree of differentiation, or type, stage of metastasis, and / or the likelihood or incidence of complications common to intermediate or late stage cancer, e.g., cancerous pleural or ascites effusion, and / or adverse effects to the subject, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response to the administered cells and / or chimeric antigen receptor.

[0285] Those skilled in the art should understand from the teachings of the present invention that the doses specifically disclosed in the present invention are safe and effective doses obtained in the research of the present inventors, and those skilled in the art, for example, clinicians, can determine the doses to be administered in each cycle based on various practical circumstances, such as factors such as the tumor burden of the patient and the patient's own physical condition, and if administration of subsequent cycles is still required, those skilled in the art can determine the doses of subsequent cycles based, for example, on the change of the tumor burden after administration of immune effector cells.

[0286] In some aspects, the tumor burden prior to administering the previous cycle of treatment determines the dose of cell therapy product administered in a subsequent cycle, in some embodiments, e.g., the previous cycle caused a decrease or reduction in tumor burden or was already below a certain threshold amount or level, e.g., above which there is an increased risk of adverse effects, indicating an increased dose in the subsequent cycle.

[0287] In some cases, the dose of the cell therapy product may be increased in subsequent cycles even if the patient's tumor burden has not decreased after receiving the previous cycle of treatment.

[0288] T cells (e.g., CAR-T cells) may be administered by any route, including intravenous injection (IV). In some embodiments, the CAR-T cells are administered by IV within the following time periods: about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes.

[0289] In some embodiments, if clinical risk indicated by biochemical indicators or markers indicative of side effects (e.g., cytokine release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, or neurotoxicity) is not present or has already passed, subsequent cycles of treatment may be administered, if necessary.

[0290] In some embodiments, whether to administer a subsequent cycle of therapy, when to administer the subsequent cycle of therapy, and / or the dose of cell therapy product administered in the subsequent cycle is determined from the presence or extent of an immune response or detectable immune response in the subject to the cells of the previous cycle or the chimeric antigen receptor expressed thereby.

[0291] In some embodiments, the time of administration of a subsequent cycle is measured from the completion of the previous cycle of cell therapy product (the day of completion of infusion of the total dose of the previous cycle is defined as day 0).

[0292] In some embodiments, a subsequent cycle of treatment is administered if the serum level of a factor indicative of CRS in the subject is not more than about 10 times, about 25 times, about 50 times, or about 100 times the serum level of that marker in the subject at a time point prior to administration of the previous cycle.

[0293] In some embodiments, a subsequent cycle of treatment is administered after a subject's outcomes associated with CRS (e.g., serum factors associated with or indicative of CRS) or clinical signs or symptoms thereof (e.g., fever, anoxia, hypotension, or neuropathy) have already reached peak levels and have begun to decline following administration of the previous cycle.

[0294] In some embodiments, the later cycle of treatment is administered when the host's adaptive immune response has not yet been detected, established, or reached a certain level, degree, or stage, hi some aspects, the later cycle of treatment is administered before the subject's memory immune response has developed.

[0295] In some embodiments, the tumor burden is monitored between the previous and subsequent cycles.

[0296] A detectable immune response refers to an amount that can be detected by any of the many known methods for evaluating specific immune responses to a particular antigen and cell. For example, in some embodiments, ELISPOT, ELISA, or cell-based antibody detection methods (e.g., by cytometry) are performed on the serum of the test subject to detect a specific type of immune response by detecting the presence of an epitope that specifically binds and / or neutralizes an antigen present on the cell, e.g., a chimeric antigen receptor-binding antibody, e.g., a CAR antibody. Some such measurements determine the isotype of the detected antibody and the type that can indicate a response and / or whether the response is a memory response.

[0297] In some aspects, the amount and extent of the immune response is detected or measured, for example, after prior or subsequent cycles of treatment.

[0298] In some aspects, disease burden is measured or detected before administration of the prior cycle of therapy, between the prior and subsequent cycles of therapy, or after administration of the subsequent cycle of therapy.

[0299] In some embodiments, after the previous cycle of treatment, the disease burden is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 100%. In some embodiments, subsequent cycles of treatment result in a further reduction in disease burden, e.g., by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100% reduction in disease burden after subsequent cycles of treatment compared to before treatment.

[0300] In some embodiments, the method improves progression-free survival or overall survival of the subject relative to other methods. For example, in some embodiments, one month after the previous cycle of treatment, the subject's progression-free survival is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, the overall survival is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject has a progression-free, recurrence-free or overall survival of at least about 1 month, or at least about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years or about 10 years. In some embodiments, the length of progression-free survival is, for example, greater than or about 1 month, or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years.

[0301] In some embodiments, treatment with the method reduces the chance of recurrence compared to other methods, for example, in some embodiments, the chance of recurrence or progression one month after the previous cycle is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less than about 6%.

[0302] Exogenous receptors expressed by cells: Cells can be exogenously modified by known biological techniques to express exogenous receptors, for example, TCR receptors, chimeric antigen receptors (CARs), T cell fusion proteins (TFPs), and T cell antigen couplers (TACs).

[0303] Exemplary exogenous receptors, e.g., sequences comprising a CAR, may comprise the amino acid sequence set forth in any of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, 52. Illustratively, a CAR of the present application comprises a sequence set forth in SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67, sequentially linked in combination with a sequence set forth in SEQ ID NOs: 80, 81, or 82, respectively. Exemplary examples include a sequence shown in SEQ ID NO: 58 linked to a sequence shown in SEQ ID NO: 80, or a sequence shown in SEQ ID NO: 58 linked to a sequence shown in SEQ ID NO: 81, and a sequence shown in SEQ ID NO: 58 linked to a sequence shown in SEQ ID NO: 82, a sequence shown in SEQ ID NO: 59 linked to a sequence shown in SEQ ID NO: 80, or a sequence shown in SEQ ID NO: 59 linked to a sequence shown in SEQ ID NO: 81, and a sequence shown in SEQ ID NO: 59 linked to a sequence shown in SEQ ID NO: 82, a sequence shown in SEQ ID NO: 60 linked to a sequence shown in SEQ ID NO: 80, or a sequence shown in SEQ ID NO: 60 linked to a sequence shown in SEQ ID NO: 81, and a sequence shown in SEQ ID NO: 60 linked to a sequence shown in SEQ ID NO: 82, a sequence shown in SEQ ID NO: 61 linked to a sequence shown in SEQ ID NO: 80, or a sequence shown in SEQ ID NO: 61 linked to a sequence shown in SEQ ID NO: 81. The sequence shown in SEQ ID NO:61 is sequentially linked to the sequence shown in SEQ ID NO:82, the sequence shown in SEQ ID NO:62 is sequentially linked to the sequence shown in SEQ ID NO:80, or the sequence shown in SEQ ID NO:62 is sequentially linked to the sequence shown in SEQ ID NO:81, and the sequence shown in SEQ ID NO:62 is sequentially linked to the sequence shown in SEQ ID NO:82, the sequence shown in SEQ ID NO:63 is sequentially linked to the sequence shown in SEQ ID NO:80, or the sequence shown in SEQ ID NO:63 is sequentially linked to the sequence shown in SEQ ID NO:81, and the sequence shown in SEQ ID NO:63 is sequentially linked to the sequence shown in SEQ ID NO:82, the sequence shown in SEQ ID NO:64 is sequentially linked to the sequence shown in SEQ ID NO:80, or the sequence shown in SEQ ID NO:64 is sequentially linked to the sequence shown in SEQ ID NO:81, and the sequence shown in SEQ ID NO:64 is sequentially linked to the sequence shown in SEQ ID NO:82, the sequence shown in SEQ ID NO:65 is sequentially linked to the sequence shown in SEQ ID NO:80,Or the sequence shown in SEQ ID NO: 65 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 65 is sequentially linked to the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 66 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 66 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 66 is sequentially linked to the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 67 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 67 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 67 is sequentially linked to the sequence shown in SEQ ID NO: 82.

[0304] Exemplarily, the CAR of the present application may be a CAR in which the sequence shown in SEQ ID NO: 70 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 70 is sequentially linked to the sequence shown in SEQ ID NO: 81 and the sequence shown in SEQ ID NO: 70 is sequentially linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 71 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 71 is sequentially linked to the sequence shown in SEQ ID NO: 81 and the sequence shown in SEQ ID NO: 71 is sequentially linked to the sequence shown in SEQ ID NO: 82, or the sequence shown in SEQ ID NO: 72 is sequentially linked to the sequence shown in SEQ ID NO: 82. the sequence shown in SEQ ID NO: 73 is linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 72 is linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 72 is linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 73 is linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 73 is linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 73 is linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 74 is linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 74 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 74 is sequentially linked to the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 75 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 75 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 75 is sequentially linked to the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 76 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 76 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 76 is sequentially linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 77 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 77 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 77 is sequentially linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 78 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 78 is sequentially linked to the sequence shown in SEQ ID NO: 81,The sequence shown in SEQ ID NO: 78 is sequentially linked to the sequence shown in SEQ ID NO: 82, the sequence shown in SEQ ID NO: 79 is sequentially linked to the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 79 is sequentially linked to the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 79 is sequentially linked to the sequence shown in SEQ ID NO: 82. Exemplary sequences are those shown in SEQ ID NO: 83, 84, or 85.

[0305] The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and refer to a polymer of amino acid residues, with no minimum length restriction. Polypeptides, including receptors and other polypeptides (e.g., linkers or peptides) provided, may contain amino acid residues, including natural and / or non-natural amino acid residues. The term further includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation. In some embodiments, the polypeptide may contain modifications to the original or native sequence, so long as the requisite activity of the protein is maintained. These modifications may be deliberate, induced, for example, by site-directed mutagenesis, or may be accidental, such as errors caused by mutations in the host producing the protein or by PCR amplification.

[0306] In the present invention, the cell expressing the exogenous receptor is generally a eukaryotic cell, e.g., a mammalian cell, and generally a human cell. In some embodiments, the cell is derived from blood, bone marrow, lymph or lymphoid organs and is a cell of the immune system (e.g., a cell of innate or adaptive immunity), e.g., a bone marrow or lymphoid cell, including lymphocytes (generally T cells, NK T cells and / or NK cells). Other exemplary cells include stem cells, e.g., multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are generally primary cells, e.g., cells isolated directly from the examinee and / or frozen from the examinee. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ T cells, CD8+ T cells and subsets thereof, such as T cells defined by function, activation state, maturation, differentiation potential, expansion, recycling, localization and / or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion status, and / or degree of differentiation. For the treated subject, the cells may be allogeneic and / or autologous. Methods for preparing cells expressing the exogenous receptor include conventional methods. In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing and / or manipulating them, and introducing them into the same subject before or after cryopreservation.

[0307] Subtypes and subsets of T cells and / or CD4+ T cells and / or CD8+ T cells include naive T (TN) cells, effector T (TEFF) cells, memory T cells and subtypes thereof, such as stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adoptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, delta / gamma T cells, or combinations thereof.

[0308] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or combinations thereof.

[0309] In some embodiments, the cells include those into which one or more nucleic acids have been introduced by genetic engineering, thereby expressing recombinant or engineered products of the nucleic acids. In some embodiments, the nucleic acids are heterologous, not generally present in the cell or sample obtained from the cell, e.g., a sample obtained from another organism or cell, or, e.g., not generally present in the cell being manipulated and / or not found in the organism from which such a cell originates. In some embodiments, the nucleic acids are not naturally occurring, e.g., the nucleic acids are not found in nature, and include chimeric combinations including nucleic acids encoding various domains from multiple different cell types, e.g., CARs, chimeric cytokine receptors.

[0310] Methods and vectors for genetic engineering: The present invention also provides methods, compositions and kits for generating genetically engineered cells expressing chimeric antigen receptors. Genetic engineering generally involves introducing nucleic acid encoding the recombinant or engineered moiety into a cell, for example by viral (e.g., lentivirus, retrovirus, AAV) transduction, transfection or transformation.

[0311] The term "engineered" refers to the comprehensive science and technology of using cell biology and molecular biology principles and methods to modify the genetic material in a cell or obtain a cellular product as desired, at the whole cell level or at the organelle level, by specific engineering techniques. In one embodiment, the manipulation refers to one or more modifications of a nucleic acid, e.g., a nucleic acid in the genome of an organism. In one embodiment, the manipulation refers to the modification, addition and / or deletion of a gene. In one embodiment, the engineered cell may refer to a cell with added, deleted and / or modified genes.

[0312] In some embodiments, gene transfer is accomplished by first stimulating cells, e.g., by combining with a stimulus that induces a response, e.g., proliferation, survival and / or activation, detected, e.g., by expression of a cytokine or activation marker, and then transducing the activated cells and expanding them in culture to sufficient quantities for clinical use.

[0313] In some aspects, the cells are engineered to enhance expression of cytokines or other factors.

[0314] In some embodiments, recombinant infectious viral particles, such as vectors derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV), are used to introduce the recombinant nucleic acid into cells. In some embodiments, recombinant lentiviral or retroviral vectors, such as gamma-retroviral vectors, are used to introduce the recombinant nucleic acid into T cells.

[0315] In some embodiments, the recombinant nucleic acid is introduced into the T cell by electroporation. In some embodiments, the recombinant nucleic acid is introduced into the T cell by translocation (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2,e74; Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection, tungsten-based biolistic bombardment (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0316] Preparation of immune effector cells: In some embodiments, the preparation of engineered modified cells includes one or more cultures and / or one or more preparation steps. Cells for introducing a nucleic acid encoding a transgenic receptor (e.g., a CAR) may be isolated from a sample (e.g., a biological sample, e.g., a sample obtained or derived from a test subject). In some embodiments, the test subject from which the cells are isolated is one suffering from a given disease or condition or in need of cell therapy or about to be subjected to cell therapy. In some embodiments, the test subject is a human in need of a particular therapeutic intervention, e.g., in need of adoptive cell therapy or immune effector cell therapy, and the cells used in the therapy have been isolated, processed and / or engineered. In some embodiments, the cells are primary cells, e.g., primary human cells. The samples include tissues, body fluids, other samples taken directly from the test subject, and samples obtained from one or more processing steps, e.g., separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing and / or incubation. The biological sample may be a sample directly derived from an organism or a processed sample. Biological samples include body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, and processed samples derived therefrom, but are not limited to.

[0317] In some embodiments, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or a product from apheresis or leukapheresis. Exemplary samples include whole blood, PBMCs, white blood cells, bone marrow, thymus, tissue biopsy specimens, tumors, leukemias, lymphomas, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy or immune effector cell therapy), samples include autologous and allogeneic samples.

[0318] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are of xenogenic origin, e.g., obtained from a mouse, rat, non-human primate, or pig.

[0319] In some embodiments, the separation of cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more substances, e.g., to remove unwanted components, enrich for certain components, lyse or remove cells that are sensitive to a particular substance. In some examples, cells are separated by one or more properties, e.g., density, adhesion properties, size, sensitivity and / or resistance to a particular component. In some examples, cells are obtained from the examinee's circulating blood, e.g., by apheresis or leukapheresis. In some aspects, the sample includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects includes cells other than red blood cells and platelets.

[0320] In some embodiments, the method includes a density-based cell separation method, for example, preparing peripheral blood mononuclear cells (PBMCs) by Percoll or Ficoll gradient centrifugation of peripheral blood or apheresis samples or leukapheresis samples with or without lysis of red blood cells.

[0321] In some embodiments, the separation method involves separating different cell types by one or more specific molecules in the cells, e.g., surface markers, e.g., by the expression or presence of surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method of separating by such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, in some aspects, the separation involves separating cells and cell populations by the expression or expression level of one or more markers (generally cell surface markers) of the cells, e.g., by incubation with an antibody or binding partner that specifically binds to such marker, followed by separating, typically by a washing step, cells that are bound to the antibody or binding partner from those cells that are not yet bound to the antibody or binding partner.

[0322] The separation does not require 100% enrichment or depletion of a particular cell population or cells expressing a particular marker.

[0323] For example, in some embodiments, specific subsets of T cells, e.g., cells positive for one or more surface markers or cells expressing high levels of one or more surface markers, e.g., CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ T cells, are isolated by positive or negative selection techniques.

[0324] For example, CD3+,CD28+ T cells may be positively selected by CD3 / CD28-linked magnetic beads (e.g., DYNA beads M-450CD3 / CD28 T cell expander).

[0325] In some embodiments, T cells are separated from the PBMC sample by negative selection against markers (e.g., CD14) expressed in non-T cells (e.g., B cells, monocytes, or other blood leukocytes). In some aspects, a CD4+ or CD8+ selection step is used to separate helper CD4+ and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subsets by positive or negative selection against markers expressed or expressed to a relatively high extent in one or more naive, memory, and / or effector T cell subsets.

[0326] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells, e.g., by positive or negative selection with surface antigens associated with the corresponding subsets. In some embodiments, enrichment for central memory T (TCM) cells improves efficacy, e.g., improves long-term survival, expansion and / or engraftment following administration, and in some aspects is particularly preferred for such subsets.

[0327] In some embodiments, the preparation method includes a freezing step, e.g., cryopreserving the cells before or after separation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, in some cases, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be utilized. One example involves using PBS or other suitable cell freezing medium with 20% DMSO and 8% human serum albumin (HAS). It is then diluted 1:1 with medium to a final concentration of DMSO and HSA of 10% and 4%, respectively. The cells are then typically frozen to -80°C or -90°C in a program-controlled cooling device at a predefined program or principle, e.g., a rate of 1°C / min, and stored in the vapor phase of a liquid nitrogen storage tank.

[0328] In some embodiments, the cells are incubated and / or cultured prior to or simultaneously with the genetic manipulation. The incubation step may include culturing, incubation, stimulating, activating, and / or growing. In some embodiments, the cells or compositions are incubated under stimulatory conditions or in the presence of stimulatory reagents. Such conditions include conditions designed to induce proliferation, reproduction, activation, and / or survival of cells in a population to simulate contact with an antigen, and / or conditions designed for genetic manipulation in the cells, e.g., introduction of a recombinant antigen receptor.

[0329] In some embodiments, the stimulatory conditions or reagents include one or more substances, e.g., a ligand capable of activating an intracellular signaling domain of the TCR complex. In some aspects, the substance initiates or triggers a TCR / CD3 intracellular signaling cascade reaction in the T cell. Such substances may include antibodies, e.g., antibodies with specificity for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, e.g., coupled to a solid support (e.g., beads), and / or one or more cytokines. Optionally, the expansion method may further include the following steps: Add anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / mL). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15 and / or IL-7 and / or IL-21, e.g., IL-2 at a concentration of at least about 10 units / mL.

[0330] In one embodiment, antigen-specific T cells, e.g., antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones may be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and ex vivo stimulating the cells with the same antigen.

[0331] Treating cancer patients with autologous T cells expressing CAR: The method of the present invention can be summarized as follows. T cells are prepared from peripheral blood mononuclear cells (PBMCs) of a human subject suffering from cancer by the "mononuclear cell isolation method", and the T cells are cultured and transduced with a viral vector encoding a chimeric antigen receptor (CAR), which specifically binds to an antigen (tumor-associated or tumor-specific antigen) expressed by tumor cells in the subject. The cells are cryopreserved in infusion medium in a single flexible freezing bag. Each is one unit dose of cells, approximately 1 x 10 6 ~ approx. 5 x 10 cells 7Each participant received a pre-injected cycle containing approximately 1 × 10 cells. 12 10 cells or less, preferably about 1 x 10 11 cells or less, more preferably about 1×10 10 5 x 10 cells or less 9 5 cells or less or approximately 2 x 10 9 Prior to injection, the cells are kept at a temperature below about -130°C or below about -175°C.

[0332] Prior to initiating cell therapy, blood is obtained from the candidate and, optionally, the levels of one or more serum factors indicative of cytokine release syndrome (CRS), e.g., tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), IL-10 or IL-6, are assessed in the serum by ELISA and / or MSD and / or CBA methods. Prior to initiating therapy, tumor burden may optionally be assessed, e.g., by measuring the size or mass of a solid tumor by PET or CT scan.

[0333] The patient is resuscitated by raising the temperature to approximately 38°C, and the patient is administered cells from the previous cycle in multiple infusions, each given intravenously (IV) as a continuous infusion over approximately 3-30 minutes.

[0334] After administration of the previous cycle, the participant undergoes a physical examination and is monitored for any symptoms of toxicity or adverse effects, such as fever, hypotension, anoxia, neuropathy, or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Optionally, one or more times after administration of the previous cycle, blood is obtained from the patient to assess the levels of serum factors indicative of CRS by methods of ELISA and / or MSD and / or CBA. The serum factor levels are compared to those obtained just prior to administration of the previous cycle. If necessary, anti-IL6 or other CRS therapy is administered to reduce symptoms of CRS.

[0335] After administration of the previous cycle, e.g., 1 week, 2 weeks, 3 weeks, and / or 4 weeks after the start of administration, the presence or absence of an anti-CAR immune response in the subject is optionally detected, e.g., by qPCR, ELISA, ELISPOT, cell-based antibody measurements, and / or mixed lymphocyte reaction.

[0336] The percentage of tumor burden reduction achieved by the previous cycle may optionally be measured one or more times after administration of the previous cycle to patients with solid tumors by scans (e.g., PET and CT scans) and / or quantified by tumor positive cells in the blood or at the tumor site.

[0337] Participants will be monitored periodically for several years after the first dose to track and measure tumor burden and / or to measure the in vivo proliferation and persistence of the administered cells by detection of CAR-expressing cells by cytometry and quantitative polymerase chain reaction (qPCR) and / or to assess the progression of the anti-CAR immune response.

[0338] The previous cycle injected into each patient was approximately 1×10 12 10 cells or less, preferably about 1 x 10 11 cells or less, more preferably about 1×10 10 cells or less, or approximately 5 x 10 9 cells or less, or 2 x 10 9 Prior to infusion, the cells are kept at a temperature below -175°C and at the time of infusion, they are resuscitated by raising the temperature to approximately 38°C.

[0339] The dose administered in the subsequent cycle is patient-specific and based on the tumor burden, the presence of an anti-CAR immune response, and the level of CRS-related outcomes. The dose administered in the subsequent cycle is approximately 1×10 12 10 cells or less, preferably about 1 x 10 11 cells or less, more preferably about 1×10 10 cells or less, more preferably about 5×10 9 5 cells or less or approximately 2 x 10 9 Less than one cell.

[0340] Analyses will be performed using descriptive statistical methods (e.g., number of cases, mean, median, standard deviation (SD), minimum, maximum). Categorical variables will be analyzed using frequency tables (frequency and percentage). All adverse events (AEs) will be classified according to the latest version of the ICH Medical Dictionary MedDRA codes, graded according to the Common Terminology Criteria for Adverse Events (CTCAE v5.0), and analyzed using frequency distributions, graphs, or other descriptive measures to calculate the number and percentage of participants with treatment-emergent adverse events (TEAEs) by organ system class, preferred term, and group. Generate CAR-T cell copy numbers including different detection time points. Calculate 95% confidence intervals for ORR and DCR using the Clopper-Pearson method. EXAMPLES

[0341] The present invention will now be further described with specific examples. It should be understood that these examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. In the following examples, the experimental methods for which no specific conditions are specified generally follow conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, or those recommended by the manufacturer.

[0342] Materials and Methods: Exemplary antigen receptors (including CARs) of the present invention and methods for genetic engineering and introducing the receptors into cells are described in, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN108884459A, CN108610420A, CN108341872A, ,CN108456250A,CN109796532A,CN108866003A,CN108853144A,CN109385403A,CN109385400A,CN109468279A,CN109503715A,CN109880803A,CN110055275A,CN110123837A,CN110438082A,CN110468105A,International Patent Application Publication Nos. WO2018006882A1, WO2015172339A8, WO2016086813A1, WO2017032293A1, WO201 7020812A1, WO2017080377A1, WO2018108106A1, WO2018 / 133877A1, WO2018 / 149358A1, WO2018 / 219299A1, WO2019 / 096261A1, WO2018 / 210279A1, WO 2019 / 024933A1, WO2020118634A1, WO2019052562A1, WO2019047932A1, WO2019109980A1, WO2019 / 141270A1, WO2019 / 149279A1, WO2019 / 170147A1, W W O2020156554A1, WO2021 / 027785A1, WO2021 / 052496A1, WO2021 / 057906A1, WO2021 / 136550A1, WO2021148019A1, WO2021232864A1, WO2016036973A1,Please refer to the entire contents of WO2016049459A1.

[0343] Illustratively, in the following examples of the present invention, the scFv of the chimeric antigen receptor can recognize the tumor antigen claudin18.2 and has the sequence shown in SEQ ID NO: 14, the chimeric antigen receptor has the sequence shown in SEQ ID NO: 11, and the CDR regions thereof are HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0344] The method of constructing CAR-T cells is as follows: First, a CAR gene is constructed, which includes, from the 5' end to the 3' end, CD8α signal peptide (base sequence as shown in SEQ ID NO: 47), scFv (base sequence as shown in SEQ ID NO: 15), CD8 hinge region (base sequence as shown in SEQ ID NO: 48), CD28 transmembrane region (base sequence as shown in SEQ ID NO: 49), CD28 intracellular signaling domain (base sequence as shown in SEQ ID NO: 50), and CD3ζ intracellular signaling domain CD3ζ (base sequence as shown in SEQ ID NO: 51) in this order. Then, the CAR gene is cloned into the plasmid PRRLSIN-cPPT.EF-1α to obtain a target plasmid containing the CAR gene. The constructed target plasmid and packaging plasmid are co-transfected into 293T cells to generate a lentiviral vector, and the lentiviral vector is transduced into T cells obtained from the peripheral blood of the patient to obtain CAR-T cells.

[0345] The intensity of antigen (e.g., claudin 18.2) positivity is defined by the intensity of immunohistochemical staining, which is partial or total cell membrane or cytoplasmic staining, and the staining intensity is divided into 0 (negative, no staining), + (weakly positive staining), ++ (positive staining), and +++ (strongly positive staining). The percentage of stained tumor cells refers to the percentage of tumor cells with each staining intensity in the total tumor cells.

[0346] Example 1: Treatment with CAR-T cells CAR-T cell therapy was administered to 12 CLDN18.2-positive gastric cancer patients who had failed treatment with PD-1 and / or PD-L1 antibodies. CLDN18.2 expression was detected using tumor tissue sections from previous surgery or biopsy, and all were CLDN18.2-positive (≥+, 10%). All patients had distant metastasis, and the status of the 12 patients was as shown in Table 1. The PD-1 and / or PD-L1 antibodies previously used by the patients were camrelizumab, pembrolizumab, sintilimab, toripalimab, and several monoclonal antibodies in clinical trials.

[0347] Prior to receiving CAR-T cells, patients underwent pretreatment using apheresis separation technology known as "mononuclear cell isolation."

[0348] PBMCs were obtained from the subjects by apheresis, and CAR-T cells were obtained by transduction and amplification of a viral vector encoding the anti-CLDN18A2 CAR. The obtained CAR-T cells were cryopreserved in a freezing solution using a freezing bag.

[0349] Prior to receiving CAR-T cells, patients were given chemotherapy or radiation therapy to undergo lymphodepletion.

[0350] Prior to administration of CAR-T cell therapy, tumor burden may optionally be assessed by measuring the size or characteristics of a solid tumor, for example, by PET or CT scan, and may also be assessed by detecting tumor markers and / or observing the occurrence and severity of tumor complications.

[0351] If the patient is required to undergo at least one cycle of CAR-T cell therapy, the subsequent cycle of CAR-T cells will be administered 4 weeks after the completion of the previous cycle of CAR-T. Each cycle of CAR T cell administration may be administered once, or two or more times by intravenous (IV) infusion, with each administration of the cell product being completed in about 3-30 minutes, preferably 5-25 minutes.

[0352] After completing administration of CAR-T cells in each cycle, the participant will undergo a physical examination and be monitored for any symptoms of toxicity or adverse effects, such as fever, hypotension, hypoxia, neuropathy, or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Testing may involve obtaining blood from the patient and assessing the levels of cytokines indicative of CRS by ELISA, and / or MSD, and / or CBA methods. If necessary, anti-IL6 therapy or other CRS therapy will be administered to reduce symptoms of CRS.

[0353] After each cycle of CAR-T cell administration is completed, for example, at 1, 2, 3, and / or 4 weeks after the start of administration, the number of CAR-T cells in the subject's body may be evaluated by qPCR, ELISA, ELISPOT, antibody measurement, etc.

[0354] The reduction in tumor burden after each cycle of treatment may be obtained by scans (e.g., PET and CT scans) and / or by quantification of antigen (e.g., claudin 18.2) positive cells in the blood or at the tumor site.

[0355] The combination of fludarabine, cyclophosphamide, and albumin-bound paclitaxel was administered to the subjects 2 to 10 days before the CAR-T cell infusion as pretreatment for CAR-T cell therapy. All 12 patients were pretreated with fludarabine, cyclophosphamide, and albumin-bound paclitaxel. Fludarabine was administered twice, and intravenously infused at 30 to 50 mg / day 6 and 5 days (D-6, -5), or 5 and 4 days (D-5, -4), or 4 and 3 days (D-4, -3) before the CAR-T cell infusion, respectively. Seven patients received 30 to 40 mg / day and five patients received 41 to 50 mg / day, with a mean daily dose of 39.8 (± 4.08) mg and a mean daily dose by body surface area of ​​25.0 (± 0.30) mg / m2. 2 Cyclophosphamide was administered twice or three times. When cyclophosphamide was administered twice, it was administered 6 days, 5 days (D-6, -5), or 5 days, 4 days (D-5, -4), or 4 days, 3 days (D-4, -3) before administration of CAR-T cells, respectively. When cyclophosphamide was administered three times, it was administered 6 days, 5 days (D-6, -5), or 5 days, 4 days (D-5, -4), or 4 days, 3 days (D-4, -3) before administration of CAR-T cells, respectively. The dose of cyclophosphamide administered to patients was 300-600 mg / day, and the amount of cyclophosphamide administered to patients was 300-400 mg / day or 401-500 mg / day. The average daily dose was 395.8 (± 47.81) mg, which was 249.6 (± 16.38) mg / m2 based on body surface area. 2 Albumin-bound paclitaxel was administered once, 3 to 5 days before administration of CAR-T cells, for example, on D-4, albumin-bound paclitaxel was administered at 90 to 200 mg / day. The mean daily dose of albumin-bound paclitaxel was 102.2 (± 8.60) mg.

[0356] Of the 12 patients, 6 patients received two cycles of cell infusion, and the median interval between the end of the first cycle of CAR-T cell infusion and the start of the second cycle of CAR-T cell infusion was 64 days.

[0357] [Table 1]

[0358] Example 2: Evaluation of Treatment In the evaluation of the treatment of the 12 subjects according to Example 1, statistical analysis was performed for PFS, DDC, DOR and OS, and descriptive analysis was performed for TTR using the Kaplan-Meier method.

[0359] Target and non-target lesions will be detected using imaging tests to assess tumor burden before and after treatment, and the number and size of target lesions will be determined. Efficacy assessment criteria will refer to Response Evaluation Criteria in Solid Tumors v1.1.

[0360] After administration of the CAR-T cells, participants are evaluated and monitored for neurotoxicity (neurological complications including confusion, aphasia, seizures, convulsions, lethargy and / or changes in mental status) with severity grading (using a grade 1-5 rating scale, e.g., Guido Cavaletti & Paola Marmiroli, Nature Reviews Neurology 6, 657-666 (December 2010)), with grade 3 (severe symptoms), grade 4 (life-threatening symptoms) or grade 5 (death) being considered serious neurotoxicity.

[0361] (1) Identification and monitoring of cytokine release syndrome (CRS) Grade 1 (mild) - not life-threatening: only systemic treatment, e.g., antipyretics and antiemetics, is required (e.g., fever, nausea, fatigue, headache, muscle pain, discomfort). Grade 2 (moderate) - moderate intervention required, responsive: inspired oxygen concentration <40%, or hypotension requiring fluids or a low-dose single vasopressor, or grade 2 organ toxicity (per CTCAE v5.0). Grade 3 (severe) - Requires aggressive intervention, responsive: Fraction of inspired oxygen ≥ 40%, or hypotension requiring high doses of a single vasopressor (e.g., norepinephrine ≥ 20 μg / kg / min, dopamine ≥ 10 μg / kg / min, phenylephrine ≥ 200 μg / kg / min, or adrenaline ≥ 10 μg / kg / min), or hypotension requiring multiple vasopressors (e.g., antidiuretic hormone plus one of the above agents, or a vasopressor combination of ≥ 20 μg / kg / min norepinephrine), or grade 3 organ toxicity or grade 4 aminotransferase increases (per CTCAE v5.0). Grade 4 (life-threatening) - Requires ventilator support or grade 4 organ toxicity (excluding aminotransferase increases). Grade 5 (Fatal)- Death.

[0362] (2) Exemplary grading criteria for neurotoxicity Grade 1 (absent or mild clinical symptoms) - mild or no clinical symptoms. Grade 2 (Moderate) - Symptoms that limit activities of daily living (ADL), such as cooking, buying vegetables or clothes, using the telephone, or managing money. Grade 3 (Severe) - Symptoms that limit activities of daily living (ADL), such as bathing, dressing or undressing, eating, using the toilet, or taking medications. Grade 4 (life-threatening) - symptoms that are life-threatening and require immediate intervention Grade 5 (Fatal)- Death.

[0363] (3) Efficacy of treatment After the first cycle of CAR-T cell treatment, the safety and efficacy of CAR-T treatment were evaluated, and the results are shown in Table 2. As shown in Table 2, regardless of the disease burden before treatment, the first cycle of treatment did not cause severe CRS or severe neurotoxicity, so the first cycle of treatment was safe. It was also observed that after the first cycle of treatment, 5 patients had stable disease (SD), 5 patients had PR, and 2 patients had progressive disease (PD), indicating that CAR-T cells can still produce obvious efficacy in patients who have used PD-1 or PD-L1 antibodies, with the ORR reaching 41.6% (5 / 12) and the disease control rate reaching 83.3% (10 / 12).

[0364] [Table 2] TIFF2024527180000003.tif122170

[0365] A second cycle of treatment was administered to some patients, and the start time of the second cycle of treatment and the efficacy after treatment are shown in Table 3.

[0366] [Table 3]

[0367] As shown in Figure 1 , 12 patients who had previously failed PD-L1 or PD-1 antibody therapy, as assessed according to RECIST 1.1 criteria, achieved a 2.5 × 10 8 ~5.0×10 8 After treatment with CAR-T cells, the target lesions of 11 patients all shrank to varying degrees, and 6 patients achieved partial remission, with an objective response rate (ORR) (95% CI) of 50% (21.09%, 78.91%) and a disease control rate (DCR) (95% CI) of 75% (42.81%, 94.51%).

[0368] The median duration of response (mDOR) of the 6 subjects who achieved an objective response was 5.4 months (1.9, NE). Among them, 1 subject who received only 1 treatment has reached a sustained remission of 6.4 months, and 1 patient had no detectable target lesions at the time of efficacy evaluation after receiving 1 treatment, and the sustained remission time has already exceeded 4 months.

[0369] Progression-free survival (PFS) and overall survival (OS): The median follow-up time from apheresis and first CAR-T cell infusion of 12 participants was calculated using the reverse Kaplan-Meier curve method, which was about 6.4 months and about 5.6 months, respectively. The mPFS (95%CI) was calculated using the KM curve method, and the mPFS of all participants receiving CAR-T cell therapy was 4.2 months (2.2, NE), and the mOS (95%CI) was calculated using the KM curve method as 7.4 months (2.6, NE). The results showed that participants could obtain more obvious clinical benefit from CAR-T therapy, and PFS and OS were obviously prolonged.

[0370] To further evaluate the effect of cells administered in the previous and subsequent cycles, the sustained survival period of CAR-T cells in the body, i.e., the period during which CAR-T cells "implanted" in the body continue to survive, is detected. After the end of the first infusion (day 0), the copy number of CAR-CLDN18 DNA contained in peripheral blood is detected by Q-PCR (the probe used is FAM-5'-CTGAGCAGCGTGACCGCCGC-3'TAMRA (SEQ ID NO: 49), the upstream primer sequence is 5'-TGGAGTGGATCGGCTACATC-3' (SEQ ID NO: 50), and the downstream primer sequence is 5'-AGTAGTAGATGGCGGTGTCG-3' (SEQ ID NO: 51)) at each follow-up time point.

[0371] The cell metabolism results of 12 subjects showed that after the first CAR-T cell infusion, the median time to peak of the copy number amplified in the body was about 7 days, and the median peak value (minimum, maximum) of the CAR copy was 7196 (3422, 39315) [copy number / μg gDNA], respectively. After the first infusion, the CAR-T cells were amplified in the body and persisted for a maximum of 203 days. No obvious dose-dependent relationship was observed. After the second cycle of cell infusion, the median time to peak of cell amplification was shortened to within 5 days, the amplitude was obviously lower than that of the first cycle, and the median C max was 1074 copies / μg gDNA.

[0372] (4) Safety All adverse events (AEs) will be classified according to the latest version of the ICH Medical Dictionary MedDRA codes, graded according to the Common Terminology Criteria for Adverse Events (CTCAE v5.0), and analyzed using frequency distributions, graphs, or other descriptive measures to calculate the number and percentage of subjects with treatment-emergent adverse events (TEAEs) by organ system class, preferred term, and group.

[0373] In this study, CRS occurred in 94.6% of participants, all of which were grade 1 to 2. Clinical symptoms were mainly fever, tachycardia, and peripheral edema, with hypotension and hypoxemia also observed, but recovery was possible with symptomatic and supportive treatment. In patients undergoing a second cycle of infusion, the incidence of AEs with abnormal hematological detection indicators was close to that after the first treatment, indicating that reinfusion does not cause cumulative toxicity and does not increase the risk to participants.

[0374] Example 3: CAR-T treatment after failure of anti-PD-1 / PD-L1 antibody treatment in gastric cancer Similar to the previous example, 12 new CLDN18.2-positive gastric cancer patients were treated with CLDN18A2-CAR-T after progression (disease progression PD according to RECIST 1.1) on PD-1 and / or PD-L1 antibody treatment. The PD-1 and / or PD-L1 antibodies used by the patients were camrelizumab, pembrolizumab, sintilimab, toripalimab, and several monoclonal antibodies in clinical trials.

[0375] Twelve new patients who had failed previous PD-L1 or PD-1 antibody therapy, assessed according to RECIST 1.1 criteria, received approximately 2.5 × 10 8 ~5.0×10 8 After receiving 10 CAR-T cells / cycle, 4 patients had SD, 6 had PR, and only 2 had PD, indicating that CAR-T cells can still produce clear efficacy in gastric cancer patients who had previously used PD-1 or PD-L1 antibodies. All CRS occurred in the study participants were grade 1-2, and there was no neurotoxicity. Clinical symptoms were mainly fever, tachycardia, and peripheral edema, with hypotension and hypoxemia also observed, and recovery was possible with symptomatic and supportive treatment.

[0376] Comprehensive statistics on the effects of receiving CLDN18A2-CAR-T treatment after progression (disease progression PD according to RECIST 1.1) with PD-1 and / or PD-L1 antibody treatment in the 12 gastric cancer patients described in Example 1 and the new 12 gastric cancer patients described in this Example showed that 12 patients achieved partial remission, with the ORR (95% CI) reaching approximately 50% (29.1%, 70.9%) and the DCR (95% CI) reaching approximately 79.2% (57.8%, 92.9%).

[0377] The mDOR for the 12 patients who achieved PR after CAR-T therapy was 6.3 months (1.9, 9.5). Of these, 6 patients achieved an overall survival of approximately 11 months or more and were still alive and in follow-up.

[0378] The mPFS (95%CI) was calculated using the KM curve method, and the mPFS (95%CI) of all participants receiving CAR-T cell therapy was 4.9 months (2.6, 7.4), and the mOS (95%CI) was calculated using the KM curve method was 6.7 months (3.7, 9.0). The results showed that participants could obtain more obvious clinical benefit from CAR-T therapy, and the PFS and OS were obviously prolonged.

[0379] The cell metabolism results of the 24 participants showed that the median time to peak copy number amplified in the body was approximately 7 days after the first CAR-T cell infusion, and the median peak CAR copy number (minimum, maximum) was 4536 (433, 39315) [copy number / μg gDNA], respectively.

[0380] Example 4: CAR-T treatment after failure of anti-PD-1 / PD-L1 antibody treatment in Krukenberg tumors As in the previous example, a patient with CLDN18.2-positive Krukenberg tumor was treated with CLDN18A2-CAR-T after progression (progressive disease according to RECIST 1.1) on PD-1 antibody (pembrolizumab), and received approximately 2.5×10 8 Treatment with CAR-T cells achieved partial response, progression-free survival was approximately 4 months, and the patient remained in stable disease. The patient experienced grade 1 CRS and no neurotoxicity.

[0381] Example 5: CAR-T treatment after failure of anti-PD-1 / PD-L1 antibody treatment in gallbladder cancer As in the previous example, patients with CLDN18.2-positive gallbladder cancer were treated with CLDN18A2-CAR-T after progression (disease progression per RECIST 1.1) on PD-1 antibody (Pembrolizumab) therapy, and received approximately 2.5 × 10 8Treatment with CAR-T cells resulted in SD, progression-free survival of approximately 7.5 months, and continued stable disease. The patient experienced grade 2 CRS and no neurotoxicity.

[0382] Example 6: CAR-T treatment after failure of anti-PD-1 / PD-L1 antibody treatment in liver cancer As in the previous example, in this example, 11 tumor antigen GPC3-positive liver cancer patients were exemplarily treated with GPC3-CAR-T cells after progression (disease progression PD according to RECIST 1.1) with PD-1 and / or PD-L1 antibody treatment. The PD-1 and / or PD-L1 antibodies used by the patients were camrelizumab, pembrolizumab, sintilimab, toripalimab, and several monoclonal antibodies in clinical trials. All of these 11 patients had previously received at least first-line standard treatment, of which one had received first-line treatment, eight had received second-line treatment, one had received fourth-line treatment, and one had received sixth-line treatment.

[0383] According to the RECIST 1.1 criteria, 11 liver cancer patients in this example received approximately 2.5 × 10 8 ~5.0×10 8 After receiving 10 CAR-T cells / cycle treatment, 5 patients had SD, 1 had PR, and 5 had PD, indicating that CAR-T cells can still produce obvious efficacy in liver cancer patients who have used PD-1 or PD-L1 antibodies, with the disease control rate reaching approximately 54.5% (6 / 11). The mean progression-free survival of the 6 patients who achieved disease control was approximately 4.4 months, of which 4 patients had an overall survival of more than 1 year, and 3 of these 4 patients are still alive and in the follow-up period.

[0384] None of the study participants developed severe uncontrollable CRS and there was no neurotoxicity. Clinical symptoms consisted mainly of fever, tachycardia, and peripheral edema, as well as hypotension and hypoxemia, which were reversible with symptomatic and supportive care.

[0385] The copy number of CAR-GPC3 DNA contained in the peripheral blood of patients was detected by Q-PCR as described in Example 2. The results showed that the median time to peak of the copy number amplified in the body was about 7 days after the first CAR-T cell infusion.

[0386] All documents mentioned in this application are incorporated herein by reference as if each document were incorporated by reference in its entirety. It should also be understood that those skilled in the art, after reading the above teachings of the present invention, may make various modifications or amendments to the present invention, and that equivalents thereof are within the scope defined by the appended claims of this application.

[0387] In the table below the sequences used in this application are given. [Table 4] TIFF2024527180000006.tif224170TIFF2024527180000007.tif224170TIFF2024527180000008.tif225170TIFF20245271800 00009.tif224170TIFF2024527180000010.tif224170TIFF2024527180000011.tif224170TIFF2024527180000012.tif123170

Claims

1. 1. Use of a cell therapy product to treat cancer in a patient who has failed a prior cancer treatment, wherein the prior treatment included treatment with an anti-PD-1 antibody and / or an anti-PD-L1 antibody, and the cell therapy product comprises immune effector cells expressing an exogenous receptor, wherein the antigen-binding domain of the exogenous receptor specifically recognizes a tumor antigen.

2. the immune effector cells are selected from T cells, NK cells, NK T cells, mast cells, macrophages, dendritic cells, CIK cells, stem cell-derived immune effector cells, or combinations thereof; Preferably, said immune effector cells are derived from natural T cells and / or T cells induced by pluripotent stem cells, More preferably, said immune effector cells are autologous / allogeneic T cells, or primary T cells; More preferably, the T cells comprise stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Treg), effector memory T cells (Tem), γδ T cells, αβ T cells or a combination thereof.

3. 2. The use according to claim 1, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell fusion protein (TFP), a T cell-antigen coupler (TAC), or a combination thereof.

4. 2. The use according to claim 1, wherein the tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Mucl, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, CD38, B7H3, claudin6, FAP, NKG2D ligand or a combination thereof, preferably the tumor antigen is selected from GPC3, CD19, BCMA, Claudin18.2 or a combination thereof.

5. The cancer comprises a solid tumor and / or a hematological tumor, preferably the solid tumor comprises a gastrointestinal tumor, more preferably the gastrointestinal tumor comprises a gastric / esophagogastric junction adenocarcinoma, gallbladder cancer, hepatic cancer, Kruckenberg tumor or a combination thereof; Preferably, the cancer is selected from Krukenberg tumor, gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, liver cancer, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), breast cancer, and the use according to claim 1.

6. the anti-PD-1 antibody / PD-L1 antibody is selected from nivolumab, pembrolizumab, cemiplimab, camrelizumab, toripalimab, sintilimab, tislelizumab, gimverelimab (GLS-010), tislelizumab, or a combination thereof; The use of claim 1, wherein the preceding treatment preferably comprises drug therapy, surgical treatment, radiation therapy or a combination thereof, and the drug therapy comprises administration of a chemical drug and / or a biological drug, and preferably the preceding treatment comprises administration of olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compounds, bevacizumab, regorafenib, anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib or a combination thereof.

7. 2. The use according to claim 1, wherein the patient suffering from cancer is treated by administering at least one cycle of a cell therapy product, preferably the patient suffering from cancer is treated by administering 1 to 3 cycles of a cell therapy product.

8. the chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, preferably, the extracellular antigen-binding domain is derived from CD28 or CD8, and the intracellular signaling domain is derived from CD137, CD27, CD28 and / or CD3ζ; Preferably, the chimeric antigen receptor (CAR) is (i) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, or CD3ζ; (ii) an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, and CD3ζ; (iii) an antigen-binding domain that specifically binds to a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory signal domain of CD137, and CD3ζ, and / or (iv) the use according to any one of claims 7 to 14, comprising an antigen-binding domain that specifically binds to a tumor antigen, CD28 or a transmembrane domain of CD8, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and CD3ζ;

9. the dose of cells in the cell therapy product administered per cycle does not exceed about 2×10 9 cells / kg, about 2×10 8 cells / kg, or about 2×10 7 cells / kg of patient body weight, or the dose of cells in the cell therapy product does not exceed about 1×10 11 cells / patient, about 1×10 10 cells / patient, about 5×10 9 cells / patient, about 2×10 9 cells / patient, or about 1×10 9 cells / patient; Preferably, the dose of cells in the cell therapy product administered per cycle is from about 1×10 cells / kg patient weight to about 2×10 cells / kg patient weight, or from about 1×10 cells / kg patient weight to about 2×10 cells / kg patient weight, or The dose of cells in the cell therapy product administered per cycle is about 1×10 cells to about 5×10 cells / patient, about 1×10 cells to about 2×10 cells / patient, or about 1×10 cells to about 1×10 cells / patient, or the dose of cells in the cell therapy product administered per cycle is about 1×10 cells to about 5×10 cells / patient, about 1×10 cells to about 2×10 cells / patient, or about 1×10 cells to about 1×10 cells / patient, or the dose of cells in the cell therapy product administered per cycle is about 2.5×10 cells to about 5×10 cells / patient.

8. The use according to claim 7.

10. administering a pretreatment prior to administering the cell therapy product in each cycle, the pretreatment comprising administering to the patient a chemical drug, a biological drug, radiation therapy, or a combination thereof; Preferably, said pre-treatment is administered 1-8 days prior to administration of said cell therapy product, preferably 2-6 days prior to administration of said cell therapy product, preferably with no more than 4 days of continuous use of chemical drugs, biological drugs, radiation therapy or combinations thereof; More preferably, the chemical drug is selected from one or at least two of cyclophosphamide, fludarabine, a tubulin inhibitor, and a pyrimidine antitumor drug, or the chemical drug comprises cyclophosphamide and fludarabine, or the chemical drug comprises cyclophosphamide, fludarabine, and a tubulin inhibitor; The use according to claim 7, further preferably, wherein the tubulin inhibitor is a taxane compound, preferably, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel, more preferably, the taxane compound is albumin-bound paclitaxel.

11. the dosage of fludarabine is about 10-50 mg / m2 / day, or about 15-40 mg / m2 / day, or about 15-30 mg / m2 / day, or about 20-30 mg / m2 / day, or about 25 mg / m2 / day, or about 30-60 mg / day, or about 30-50 mg / day, or about 35-45 mg / day; the dosage of cyclophosphamide is about 200-400 mg / m2 / day, or about 200-300 mg / m2 / day, or about 250 mg / m2 / day, or about 300-700 mg / day, or about 300-550 mg / day, or about 300-500 mg / day; the dosage of the taxane compound is about 300 mg / day or less, or about 200 mg / day or less, or about 90-120 mg / day; Preferably, the cyclophosphamide is administered 2-3 times, or the fludarabine is administered 1-2 times, or the taxane compound is administered once. The use according to claim 10.

12. The antigen-binding domain comprises: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6, or HCDR1 as shown in SEQ ID NO: 16, HCDR2 as shown in SEQ ID NO: 17, HCDR3 as shown in SEQ ID NO: 18, LCDR1 as shown in SEQ ID NO: 19, LCDR2 as shown in SEQ ID NO: 20, LCDR3 as shown in SEQ ID NO: 21, or It has HCDR1 shown in SEQ ID NO: 27, HCDR2 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, and LCDR3 shown in SEQ ID NO: 32, or the antigen-binding domain is a heavy chain variable region shown in SEQ ID NO:7 or a heavy chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:9 or a light chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:9, or a heavy chain variable region shown in SEQ ID NO:22 or a heavy chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:22 and a light chain variable region shown in SEQ ID NO:23 or a light chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:23, or a heavy chain variable region shown in SEQ ID NO:33 or a heavy chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:33 and a light chain variable region shown in SEQ ID NO:34 or a light chain variable region having 90% or more sequence identity to the sequence shown in SEQ ID NO:34, or the antigen-binding domain comprises a sequence as set forth in SEQ ID NO: 14, 24, 35, or 37, or a sequence with 90% or more sequence identity to the sequence as set forth in SEQ ID NO: 14, 24, 35, or 37.

13. The chimeric antigen receptor comprises an amino acid sequence shown in any one of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, 52, 83, 84, and 85, or a sequence having 90% or more sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, 52, 83, 84, and 85, or a sequence shown in any one of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67, or a sequence having 90% or more sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67, respectively, The use according to claim 3, comprising a polypeptide comprising a sequence sequentially linked and combined with a sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO: 80, 81 or 82, or a polypeptide comprising a sequence sequentially linked and combined with a sequence shown in any of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79, or a sequence having 90% or more sequence identity to the amino acid sequence shown in any of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79, respectively, and a sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO: 80, 81 or 82, respectively.

14. assessing the patient's serum levels of cytokines indicative of CRS, cytokines indicative of neurotoxicity, indicators of tumor burden, and / or factors indicative of a host anti-CAR immune response prior to administering the cell therapy product in each cycle; or 8. The use of claim 7, wherein after treatment with the cell therapy product, the patient does not exhibit severe CRS or neurotoxicity above grade 3.

15. at least a portion of patients who have failed cancer treatment with the anti-PD-1 antibody or anti-PD-L1 antibody, preferably at least 40%, more preferably at least 50%, respond to the cell therapy product; 2. The use according to claim 1, wherein the response is an ORR of greater than 40%, preferably greater than 50%.

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