Improvement of the effectiveness and lasting response of immunotherapy

Abstract

This specification provides methods for preparing, producing, processing, culturing, isolating, or creating cells suitable for immunotherapy or cell therapy, and for using them in cell therapy. It further provides methods for treating cancer patients using such cells.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 381,525, entitled “Efficacy and Durable Response of Immunotherapy,” filed on 28 October 2022; U.S. Provisional Patent Application No. 63 / 386,457, entitled “Efficacy and Durable Response of Immunotherapy,” filed on 7 December 2022; U.S. Provisional Patent Application No. 63 / 479,877, entitled “Efficacy and Durable Response of Immunotherapy,” filed on 13 January 2023; and U.S. Provisional Patent Application No. 63 / 515,492, entitled “Efficacy and Durable Response of Immunotherapy,” filed on 25 July 2023, all of which are incorporated herein by reference.

[0002] This application relates to CAR-T cells, methods for producing them, and methods for using them to treat cancer. [Background technology]

[0003] Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformations to become abnormal cancer cells. Cancer cells express proteins and other antigens different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells such as T lymphocytes and B lymphocytes from normally targeting them. Human T-cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in a target, e.g., a patient. Various techniques have been developed to generate populations of chimeric antigen receptor (CAR)-T cells for cancer therapy by preparing T cell populations rich in naturally occurring T cells capable of targeting tumor antigens, removing circulating tumor cells, and / or genetically modifying T cells to specifically target known cancer antigens. Some of these therapies have shown promising effects on tumor size and patient survival rates. There is a need for cancer treatment methods that utilize (CAR)-T cells, including methods that involve bridging therapy. [Overview of the project]

[0004] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. While the present invention is described in conjunction with its detailed description, this description is illustrative and does not limit the scope of the invention as partially defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following embodiments / claims.

[0005] 1. A method for treating mantle cell lymphoma (MCL) or B-cell ALL in a subject requiring treatment for mantle cell lymphoma (MCL) or B-cell ALL, comprising administering a therapeutically effective dose of a T-cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR) to the subject, wherein the MCL or B-cell ALL is relapsed or refractory MCL after one or more prior treatments selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation (SCT), or any combination thereof, and further comprising one or more prior treatments that do not include Bruton's tyrosine kinase inhibitors (BTKi).

[0006] 2. The method according to embodiment 1, wherein the subject has received 1 to 5 types of prior treatments, and at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibody, and / or chemotherapy including anthracycline or bendamustine.

[0007] 3. The method according to embodiment 1 or 2, wherein the BTKi is ibrutinib or acalabrutinib.

[0008] 4. The method according to any one of embodiments 1 to 3, wherein R / RB cell ALL is defined as resistance to first-line therapy (i.e., resistance to primary treatment), relapse within 12 months of initial remission, relapse or resistance after two or more prior systemic therapies, or relapse after allogeneic SCT, and the subjects are required to have a myeloblast percentage of 5% or more, an Eastern Cooperative Oncology Group performance status of 0 or 1, and / or sufficient renal, hepatic, and cardiac function.

[0009] 5. The method according to any one of embodiments 1 to 4, wherein, if the subject of B-cell ALL has been previously administered blinatumomab, the subject is required to have leukemic blasts with CD19 expression of ≥90%.

[0010] 6. The method according to any one of embodiments 1 to 5, wherein the subject receives bridging therapy after leukocyte apheresis and before pre-treatment / lymphocyte apheresis chemotherapy.

[0011] 7. The subject of the above MCL is receiving intravenous cyclophosphamide 500 mg / m². 2 and intravenous fludarabine 30 mg / m² 2 The method according to any one of embodiments 1 to 6, wherein the patient receives a lymphocyte-depleting chemotherapy regimen, both of which are administered 5 days, 4 days, and 3 days prior to T-cell infusion, respectively.

[0012] 8. The B-cell ALL subjects received intravenous (IV) fludarabine 25 mg / m² 4, 3, and 2 days prior to T-cell injection. 2 / day, and IV cyclophosphamide 900mg / m² two days before the infusion. 2 The method according to any one of embodiments 1 to 7, wherein the patient undergoes a lymphocyte removal regimen of / day.

[0013] 9. The method according to any one of embodiment 6 or 8, wherein the MCL bridging therapy is selected from dexamethasone (e.g., PO or IV 20-40 mg equivalent, once daily for 1-4 days); methylprednisolone, ibrutinib (e.g., PO 560 mg, once daily), and / or acalabrutinib (e.g., PO 100 mg, twice daily); immunomodulators; R-CHOP, bendamustine; alkylating agents; and / or platinum-based agents, and the bridging therapy is administered after leukocyte apheresis and completed, for example, within 5 days prior to pre-treatment chemotherapy.

[0014] 10. The method according to any one of embodiments 6 to 8, wherein the subject of B cell ALL may receive one or more of the following bridging chemotherapy regimens. TIFF2026508791000001.tif89170

[0015] 11. The method according to any one of embodiments 1 to 10, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

[0016] 12. The method according to embodiment 11, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3ζ domain.

[0017] 13. The method according to embodiment 11 or 12, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from leukocyte apheresis-derived products in which positive selection for CD4+ and CD8+ T cells has not been performed.

[0018] 14. The method according to any one of embodiments 11 to 13, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from a leukocyte apheresis-derived product that has not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

[0019] 15. The method according to embodiment 14, wherein the superior product characteristics are selected from an increase in the proportion of CDRA45+CCR7+(naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and / or a decrease in the proportion of CD3- cells.

[0020] 16. The target of the above MCL is 1.8 × 10 per kg of body weight. 6 , 1.9 × 10 6 , or 2 × 10 6 A maximum of 2 × 10⁶ CAR-positive live T cells. 8 Patients receiving one or more CAR-positive live T cells (for patients weighing 100 kg or more) are subject to the B cell ALL regimen, which is 0.5 × 10⁶ per kg of body weight.6 、1×10 6 、 or 2×10 6 viable CAR-positive T cells, up to 2×10 8 viable CAR-positive T cells (for patients over 100 kg) are administered, according to any one of embodiments 1 to 15.

[0021] 17. If the subject achieves a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells, provided that there is no suspicion of neutralizing antibodies against the CAR and CD19 expression is maintained when the disease progresses after remission for more than 3 months. The response is evaluated using the Lugano classification, according to any one of embodiments 1 to 15.

[0022] 18. After T cell administration, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity, according to any one of embodiments 1 to 17.

[0023] 19. The subject is monitored daily for at least 7 days, preferably every 4 weeks, after infusion for signs and symptoms of CRS and neurotoxicity, according to the method of embodiment 18.

[0024] 20. The signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxemia, and / or hypotension, and the signs or symptoms associated with neurological events include encephalopathy, convulsions, changes in level of consciousness, speech disorders, tremors, and / or delirium, according to the method of embodiment 18 or 19.

[0025] 21. Cytokine release syndrome in subjects with MCL is managed according to the following protocol, according to any one of embodiments 18 to 20. TIFF2026508791000002.tif153170

[0026] 22. Neurotoxicity in subjects with MCL is managed according to the following protocol, according to any one of embodiments 18 to 21. TIFF2026508791000003.tif138170

[0027] 23. The method according to any one of embodiments 1 to 22, wherein the subject of the MCL is a high-risk patient determined by a Ki-67 tumor growth index of 50% or more and / or the presence of a TP53 mutation.

[0028] 24. The method according to any one of embodiments 18 to 20, wherein CRS in a subject of B-cell ALL is managed according to the following protocol. TIFF2026508791000004.tif140170

[0029] 25. The method according to any one of embodiments 18-20 and 24, wherein neurotoxicity in subjects with B cell ALL is controlled according to the following protocol. TIFF2026508791000005.tif244170

[0030] 26. The method according to any one of embodiments 1 to 25, wherein the subject of B cell ALL may receive one or more of the following bridging chemotherapy regimens. TIFF2026508791000006.tif85170

[0031] 27. Autologous T cells expressing anti-CD19 CAR for use in a method for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 26.

[0032] 28. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 26.

[0033] 29. A method for treating mantle cell lymphoma (MCL) in a subject requiring treatment for the mantle cell lymphoma (MCL), comprising administering to the subject a therapeutically effective amount of a T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), wherein the MCL is relapsed or refractory MCL, and the last prior therapy occurred less than 60 months prior to the administration of the T cell product.

[0034] 30. The method according to aspect 29, wherein the MCL is resistant to one or more of the following: chemotherapy, radiotherapy, immunotherapy (including T-cell therapy and / or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof, or subsequently relapses.

[0035] 31. The method according to embodiment 29 or 30, wherein the subject has received one to three prior treatments, and at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibody, anthracycline or bendamustine chemotherapy, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

[0036] 32. The method according to embodiment 31, wherein BTKi is ibrutinib.

[0037] 33. The method according to embodiment 32, wherein ibrutinib was the last treatment administered before the administration of the T-cell product.

[0038] 34. The method according to any one of embodiments 29 to 33, wherein the subject has undergone leukocyte apheresis and has not received bridging therapy prior to preconditioning / lymphocyte apheresis chemotherapy.

[0039] 35. The method according to any one of embodiments 29 to 34, wherein the subject has not undergone prior platinum therapy.

[0040] 36. The subject receiving intravenous cyclophosphamide 500 mg / m² 2 and intravenous fludarabine 30 mg / m² 2The method according to any one of embodiments 29 to 35, wherein the patient receives a lymphocyte depletion chemotherapy regimen, both of which are administered 5 days, 4 days, and 3 days prior to T cell infusion, respectively.

[0041] 37. The method according to any one of embodiments 29 to 36, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

[0042] 38. The method according to embodiment 37, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3ζ domain.

[0043] 39. The method according to embodiment 37 or 38, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from leukocyte apheresis-derived products in which positive selection for CD4+ and CD8+ T cells has not been performed.

[0044] 40. The method according to any one of embodiments 37 to 39, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from leukocyte apheresis-derived products that have not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

[0045] 41. The method according to embodiment 40, wherein the superior product characteristics are selected from an increase in the proportion of CDRA45+CCR7+(naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and a decrease in the proportion of CD3- cells.

[0046] 42. For the above subjects, 1.8 × 10 per kg of body weight 6 pieces, 1.9×10 6 individual or 2 × 106 One or more doses of CAR-positive live T cells are administered, up to a maximum of 2 × 10⁶ 8 The method according to any one of embodiments 29 to 41, wherein 100 CAR-positive live T cells (in the case of a patient weighing 100 kg or more) are administered.

[0047] 43. The method according to any one of embodiments 29 to 42, wherein if the subject achieves a complete response to the initial injection, the subject may receive a second injection of anti-CD19 CAR T cells, provided that if the condition worsens after more than three months of remission, CD19 expression is maintained and there is no suspicion of neutralizing antibodies against CAR, and the response is evaluated using the Lugano classification.

[0048] 44. The method according to any one of embodiments 29 to 43, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity after T cell administration.

[0049] 45. The method according to embodiment 44, wherein the subject is monitored daily for at least 7 days, preferably 4 weeks, after injection for signs and symptoms of CRS and neurotoxicity.

[0050] 46. ​​The method according to any one of embodiments 44 and 45, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and / or hypotension, and the signs or symptoms associated with neurotoxicity include encephalopathy, seizures, altered level of consciousness, speech disorder, tremor, and / or confusion.

[0051] 47. The method according to any one of embodiments 44 to 46, wherein cytokine release syndrome in a subject of MCL is managed according to the following protocol. TIFF2026508791000007.tif139170

[0052] 48. The method according to any one of embodiments 44 to 47, wherein neurotoxicity in subjects with MCL is controlled according to the following protocol. TIFF2026508791000008.tif135170

[0053] 49. The method according to any one of embodiments 29 to 48, wherein the subject is a high-risk patient determined by a Ki-67 tumor growth index of 50% or more and / or the presence of a TP53 mutation.

[0054] 50. Autologous T cells expressing anti-CD19 CAR for use in a method for treating MCL according to any one of embodiments 29 to 49.

[0055] 51. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating MCL according to any one of embodiments 29 to 50.

[0056] 52. A method for treating a cancer selected from the group consisting of Waldenström macroglobulinemia, Richter transformation, Burkitt lymphoma, and pilaris cell leukemia in a subject requiring treatment for such cancer, comprising administering to the subject a T-cell product containing autologous T cells expressing an effective dose of anti-CD19 chimeric antigen receptor (CAR), wherein the subject receives bridging therapy after leukocyte apheresis and before pre-treatment / lymphocyte apheresis chemotherapy.

[0057] 53. The method according to aspect 52, wherein the cancer is refractory to one or more of the following: chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation, or any combination thereof, or has subsequently relapsed.

[0058] 54. The method according to embodiment 52 or 53, wherein the bridging therapy is completed at least 7 days or 5 half-lives before the pre-treatment chemotherapy.

[0059] 55. The subject receiving intravenous cyclophosphamide 500 mg / m² 2 and intravenous fludarabine 30 mg / m² 2The method according to any one of embodiments 52 to 54, wherein the patient receives a lymphocyte-depleting chemotherapy regimen, both of which are administered 5 days, 4 days, and 3 days prior to T-cell infusion, respectively.

[0060] 56. The method according to any one of embodiments 52 to 55, wherein the cancer is Richter transformation, and the bridging therapy is selected from the group consisting of rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine and prednisone (R-CHOP); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R); Bruton's tyrosine kinase inhibitor (BTKi) (BTKi) ± VTX-2337; dexamethasone; and irradiation.

[0061] 57. The method according to any one of embodiments 52 to 55, wherein the cancer is Burkitt lymphoma, and the bridging therapy is selected from the group consisting of rituximab, ifosfamide, carboplatin and etoposide (R-ICE); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R); rituximab, gemcitabine and oxaliplatin (R-GEMOX); cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride and dexamethasone (HyperCVAD); dexamethasone; and irradiation.

[0062] 58. The method according to any one of embodiments 52 to 55, wherein the cancer is Waldenström macroglobulinemia and the bridging therapy is ibrutinib.

[0063] 59. The method according to any one of embodiments 52 to 58, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

[0064] 60. The method according to embodiment 59, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3ζ domain.

[0065] 61. The method according to embodiment 59 or 60, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from leukocyte apheresis-derived products in which positive selection for CD4+ and CD8+ T cells has not been performed.

[0066] 62. The method according to any one of embodiments 59 to 61, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from a leukocyte apheresis-derived product that has not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

[0067] 63. The method according to embodiment 62, wherein the superior product characteristics are selected from an increase in the proportion of CDRA45+CCR7+(naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and a decrease in the proportion of CD3- cells.

[0068] 64. For the above subjects, 1.8 × 10 per kg of body weight 6 pieces, 1.9×10 6 individual or 2 × 10 6 One or more doses of CAR-positive live T cells are administered, up to a maximum of 2 × 10⁶ 8 The method according to any one of embodiments 52 to 63, wherein 100 CAR-positive live T cells (in the case of a patient weighing 100 kg or more) are administered.

[0069] 65. The method according to any one of embodiments 52 to 64, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity after T cell administration.

[0070] 66. The method according to embodiment 65, wherein the subject is monitored daily for at least 7 days, preferably 4 weeks, after injection for signs and symptoms of CRS and neurotoxicity.

[0071] 67. The method according to aspect 65 or 66, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and the signs or symptoms associated with neurological events include encephalopathy, seizures, altered level of consciousness, speech disorders, tremors, and confusion.

[0072] 68. Autologous T cells expressing anti-CD19 CAR for use in a method for treating cancer according to any one of embodiments 52 to 67.

[0073] 69. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating cancer according to any one of embodiments 52 to 67.

[0074] 70. A method for treating cancer in a subject requiring cancer treatment, wherein the subject has been previously administered a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), and a peripheral blood sample has been taken from the subject after administration of the first T cell product, and the method comprises: (a) measuring the level of CD8+CD27-CD28+ T cells in the blood sample; and (b) if the level of CD8+CD27-CD28+ T cells in the blood sample is elevated, administering a second T cell product to the subject.

[0075] 71. A method for treating cancer in a subject requiring cancer treatment, wherein the subject has previously been administered a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), and a peripheral blood sample has been taken from the subject after administration of the first T cell product, and the method comprises: (a) measuring the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample; and (b) if the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample is elevated, administering a second T cell product to the subject.

[0076] 72. The method according to any one of embodiment 70 or 71, wherein the first T cell product comprises CD4+ and CD8+ T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

[0077] 73. The method according to embodiment 72, wherein the CD4+ and CD8+ T cells are activated with an anti-CD3 antibody and an anti-CD28 antibody in the presence of IL-2, and then transduced with a non-replicating viral vector encoding a chimeric antigen receptor (CAR) containing an anti-CD19 single-strand variable fragment (scFv), CD28 and CD3 zeta domains.

[0078] 74. The method according to any one of embodiments 70 to 73, wherein the cancer is selected from the group consisting of mantle cell lymphoma (MCL), B-cell ALL, Waldenström macroglobulinemia, Richter transformation, Burkitt lymphoma, and pilaris cell leukemia.

[0079] 75. The method according to embodiment 74, wherein the cancer is MCL.

[0080] 76. The method according to any one of embodiments 70 to 75, wherein the blood sample is collected from the subject between the 5th and 9th day after administration of the first T cell product.

[0081] 77. The method according to embodiment 76, wherein the blood sample is collected from the subject between day 6 and day 8 after administration of the first T cell product.

[0082] 78. The method according to embodiment 77, wherein the blood sample is collected from the subject seven days after administration of the first T cell product.

[0083] 79. The method according to any one of embodiments 70 to 75, wherein the blood sample is collected from the subject between 12 and 16 days after administration of the first T cell product.

[0084] 80. The method according to embodiment 79, wherein the blood sample is collected from the subject between 13 and 15 days after administration of the first T cell product.

[0085] 81. The method according to embodiment 80, wherein the blood sample is collected from the subject 14 days after administration of the first T cell product.

[0086] 82. The method according to embodiment 70, wherein the elevated level of CD8+CD27-CD28+ T cells in the subject is determined by comparison with other subjects who have been administered an equivalent T cell product and who have had peripheral blood samples taken on the same day after administration of the T cell product.

[0087] 83. The method according to embodiment 71, wherein the elevated level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the subject is determined by comparison with other subjects who have been administered an equivalent T cell product and whose peripheral blood samples were collected on the same day after administration of the T cell product.

[0088] 84. The method according to any one of embodiments 70 to 83, wherein the second T cell product is selected from the group consisting of autologous CD19 / CD20 bisistronic T cell products and allogeneic T cell products.

[0089] 85. A T-cell product for use in a method for treating cancer according to any one of embodiments 70 to 84.

[0090] 86. Use of a T-cell product in the manufacture of a pharmaceutical product for treating cancer according to any one of embodiments 70 to 84.

[0091] 87. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administration of the first T cell product, (b) Measuring the level of CD8+CD27-CD28+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of CD8+CD27-CD28+ T cells in the blood sample, wherein if the level of CD8+CD27-CD28+ T cells is elevated, a second T cell product is administered.

[0092] 88. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administration of the first T cell product, (b) Measuring the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, wherein if the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells is elevated, a second T cell product is administered.

[0093] 89. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administration of the first T cell product, (b) Measuring the level of CD27+CD28-CD4+CD3+ T cells in the blood sample, (c) A method for prescribing a series of treatments based on the level of CD27+CD28-CD4+CD3+ T cells in the blood sample, wherein if the level of CD27+CD28-CD4+CD3+ T cells is elevated, the second T cell product is not administered.

[0094] 90. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administration of the first T cell product, (b) Measuring the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells in the blood sample, (c) A method for prescribing a series of treatments based on the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells in the blood sample, wherein if the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells is elevated, the series of treatments is prescribed without administering a second T cell product. [Brief explanation of the drawing]

[0095] [Figure 1] The ZUMA-2 study design is shown. Superscripts are defined as follows: a. Administered after leukocyte apheresis and completed at least 5 days before initiating pre-conditioning chemotherapy; PET-CT was required after bridging; b. Bone marrow biopsy was performed at screening, and if positive, not performed, or inconclusive, biopsy was required to confirm complete response (CR); c. Only targeted adverse events (neurological, hematological, infectious, GVHD, autoimmune disorders, and secondary malignancies) were monitored and reported for 15 years, until 3 months later, after the first anti-CD19 CAR T cell infusion, or until disease progression or initiation of subsequent anticancer therapy occurred first.

[0096] [Figure 2]The following describes the treatment of patients based on their response at 24 months: Complete response (CR), Partial response (PR). [Modes for carrying out the invention]

[0097] Unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout the application. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, the Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of the terms used in this application.

[0098] Units, prefixes, and symbols are presented in the format accepted by the Systeme International de Unites (SI). Numerical ranges include the number defining the range. Disclosures provided herein may be limited by reference to the entire specification rather than by any limitation of any particular aspect of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to whom this disclosure relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology," 5th ed., (2013), Academic Press; and "The Oxford Dictionary of Biochemistry and Molecular Biology," Cammack et al. eds., 2nd ed., (2006), Oxford University Press, provide those skilled in the art with many common dictionaries of the terms used herein.

[0099] The article "a" or "an" refers to "one or more" of any cited or enumerated elements.

[0100] The terms “about” or “essentially include” refer to a value or composition that falls within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this depends to some extent on how that value or composition is measured or determined, i.e., on the limits of the measurement system. For example, “about” or “essentially include” may mean that it is within a range of 1 × or more standard deviations by practice in the art. Alternatively, “about” or “essentially include” may mean a range of up to 10% (i.e., ±10%). For example, about 3 mg may include any number between 2.7 mg and 3.3 mg (in the case of 10%). With respect to biological systems or processes, this term may mean up to one order of magnitude of the value, or up to five times. Where a particular value or composition is presented in this application, unless otherwise specified, the meaning of “about” or “essentially include” is that it includes an acceptable margin of error for that value or composition. Any range of concentration, percentage, ratio, or integer includes any integer value within the listed range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0101] Where used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.” The term “and / or” refers to each of two specified features or components, with or without the other. Thus, in this specification, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone), B (alone), and C (alone).

[0102] The terms “for example” and “that is” are used merely as examples and are not intended to be limiting, and should not be construed as referring only to the items explicitly listed herein.

[0103] Terms like "greater than or equal to," "at least," and "greater than," for example, "at least one," are not limiting, but they mean at least 1, 2, 3, 4, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 6 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, This includes 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or values ​​greater than the listed values. It also includes any larger numbers or fractions in between. The term "less than or equal to" includes each value less than the listed values.For example, "100 or fewer nucleotides" include 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 5 This includes 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any fewer number or fraction in between is also included.

[0104] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limiting, but they mean at least 2, 3, 4, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 10 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 , including 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

[0105] Throughout this specification, the word “comprising” or variations such as “comprises” or “comprising” is understood to mean including the elements, integers, or steps, or groups of elements, integers, or steps described, but not to mean excluding any other elements, integers, or steps, or groups of elements, integers, or steps. Whenever an aspect is described herein using the word “comprising,” it is understood that other similar aspects described using the terms “consisting of” and / or “consisting essentially of” are also presented. The term “consisting of” excludes any elements, processes, or components not specified in the claims. In re Gray, 53 F.2d 520,11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448,450 (Bd.App.1948) ("consisting of" is defined as "closing the claim to the inclusion of materials other than those described, excluding impurities that are usually associated with them"). The term "essentially consisting of" limits the claim to the identified materials or processes, and those that "do not substantially affect the fundamental and novel features" of the claimed patent.

[0106] As used herein, unless otherwise specified or evident from the context, the term “about” means a value or composition that is within the acceptable margin of error of a particular value or composition as determined by those skilled in the art, and this depends to some extent on how such value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “about” or “approximately” may mean within one or two standard deviations or more, according to convention in the art. “About” or “approximately” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may encompass any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, this term may mean up to an order of magnitude off or up to five times the value. Where specific values ​​or compositions are presented in this disclosure, unless otherwise specified, the meaning of “approximately” or “about” should be assumed to be within the acceptable margin of error for those specific values ​​or compositions.

[0107] As described herein, any range of concentration, percentage, ratio, or integer should be understood to include any integer values ​​within the listed ranges, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0108] The terms “activated” and “activated” refer to a cellular state, not limited to immune cells (e.g., T cells), that is sufficiently stimulated to induce detectable cell proliferation. Activation may be associated with induced cytokine production and detectable effector function. The term “activated T cell” refers, in particular, to T cells undergoing cell differentiation. T cell activation may be characterized by increased T cell expression of one or more biomarkers, including but not limited to CD57, PD1, CD107a, CD25, CD137, CD69, and / or CD71. Methods for activating and proliferating T cells are known in the art, for example, described in U.S. Patents 6,905,874, 6,867,041, and 6,797,514, and International Publication No. 2012 / 079000, the contents of which are incorporated herein by reference in their entirety. Generally, such methods involve contacting cells (such as T cells) with activators, stimulants, or co-stimulants (such as anti-CD3 and / or anti-CD28 antibodies) that can be attached to, coated, or bound to beads or other surfaces in a solution (such as a supply medium, culture medium, and / or growth medium) containing specific cytokines (such as IL-2, IL-7, and / or IL-15). The activator (such as anti-CD3 and / or anti-CD28 antibodies) attached to the same beads functions as a "surrogate" antigen-presenting cell (APC). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulant system for the physiological activation of human T cells. In one embodiment, T cells are activated and stimulated to proliferate with specific antibodies and / or cytokines using a method described in U.S. Patents 6,040,177 and 5,827,642 and International Publication No. 2012 / 129514, the entire contents of which are incorporated herein by reference.

[0109] The terms “administer,” “to administer,” etc., refer to the physical delivery of a drug to a target using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of immune cells prepared by the methods disclosed herein include, for example, intravenous (iv or IV), intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion. Parenteral administration refers to administration methods other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In one embodiment, immune cells (e.g., T cells) prepared by the method are administered by injection or infusion. Non-parenteral routes of administration include topical, cutaneous, or mucosal routes, such as intranasal, intravaginal, rectal, sublingual, or topical administration. Administration may also be carried out once, twice, or multiple times over one or more periods. When one or more therapeutic agents (e.g., cells) are administered, the administration may be carried out simultaneously or sequentially. Sequential administration includes administering one agent only after the administration of one or more other agents has been completed.

[0110] The term “antibody” (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains, CH1, CH2, CH3, and / or CH4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions referred to as “complementarity-determining regions” (CDRs), with more conservative regions referred to as “framework regions” (FRs) interposed between them. Each VH and VL contains three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. Immunoglobulins may originate from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG subclass is also well known to those skilled in the art and includes, but is not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab class or subclass encoded by a heavy chain constant region gene (e.g., IgM or IgG1). The term "antibody" includes, by example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, totally synthetic antibodies, and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods that reduce their immunogenicity in humans. Unless otherwise explicitly stated or indicated by the context, the term “antibody” includes any of the aforementioned immunoglobulins, including antigen-binding fragments or antigen-binding moieties, monovalent and bivalent fragments or moieties, and single-chain antibodies.

[0111] "Antigen-binding molecule," "antibody fragment," etc., refer to any part of an antibody smaller than the whole. Antigen-binding molecules may include the antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. In one embodiment, a CD19 CAR construct contains an anti-CD19 single-strand FV. A "single-strand Fv" or "scFv" antibody-binding fragment contains the variable heavy chain (V) of the antibody. H ) domain and variable light chain (V L ) contains domains, and these domains are present in a single polypeptide chain. Generally speaking, Fv polypeptides are V H Domain and V L The scFv further comprises a polypeptide linker between the domains, thereby enabling the scFv to form a desired structure for antigen binding. All antibody-related terms used herein have their conventional meanings in the art and are well understood by those skilled in the art.

[0112] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may involve either antibody production or activation of specific immune cells, or both. Those skilled in the art will readily understand that virtually all proteins or peptides, and any macromolecule, can function as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or by recombination. Antigens may be specific to certain tissues, such as cancer cells, or they may be expressed broadly. Furthermore, larger molecular fragments may act as antigens. In some embodiments, the antigen is a tumor antigen.

[0113] The term "neutralize" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or alters the ligand's ability to bind through indirect means (such as structural or energetic changes to the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0114] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the method of engineered autologous cell therapy described herein involves collecting lymphocytes from an individual (such as a donor or patient), which are then engineered to express a CAR construct, and subsequently administered to the same individual.

[0115] The term "allogeneic" refers to any material that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

[0116] The term "bridging therapy" refers to treatment performed between apheresis / leukocyte apheresis and the initiation of lymphocyte depletion / pre-conditioning chemotherapy.

[0117] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Unregulated cell division and proliferation lead to the formation of malignant tumors, which may invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors of various stages. In one embodiment, the cancer or tumor is stage 0, for example, the cancer or tumor is in the very early stages of development and has not metastasized. In another embodiment, the cancer or tumor is stage I, for example, the cancer or tumor is relatively small in size, has not spread to nearby tissues, and has not metastasized. In yet another embodiment, the cancer or tumor is stage II or stage III, for example, the cancer or tumor is larger than stage 0 or stage I, has grown into adjacent tissues, but has not metastasized except possibly to lymph nodes. In an additional embodiment, the cancer or tumor is stage IV, for example, the cancer or tumor has metastasized. Stage IV may also be called advanced or metastatic cancer.

[0118] As used herein, “antitumor effect” refers to a biological effect that may be presented in no particular order, including but not limited to a reduction in tumor volume, inhibition of tumor growth, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number / degree of metastases, an increase in overall survival or progression-free survival, an extension of life expectancy, and / or improvement of various physiological symptoms associated with tumor. An antitumor effect may also refer to the prevention of tumor development, for example, by a vaccine.

[0119] The term "progression-free survival" (PFS) refers to the period from the date of treatment to the date of disease progression (according to general guidelines such as the revised IWG response criteria for malignant lymphoma) or death from any cause. The term "disease progression" may be assessed by measurement of malignant lesions by radiography or other methods and should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms may be reported as the primary tumor type (e.g., DLBCL). The term "duration of response" (DOR) refers to the period from the first objective response of the subject to the date of confirmed disease progression (according to general guidelines such as the revised IWG response criteria for malignant lymphoma) or death. The term "overall survival" (OS) refers to the period from the date of treatment to the date of death.

[0120] "Cytokines" refer to non-antibody proteins that can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. In one embodiment, one or more cytokines are released in response to treatment. In other embodiments, those cytokines secreted in response to treatment may indicate or suggest an effective therapeutic agent. In one embodiment, "cytokines" refer to non-antibody proteins released by one cell in response to contact with a specific antigen, and the cytokines interact with a second cell to mediate a response in the second cell. As used herein, "cytokines" means proteins released by a population of cells that act on another cell as intercellular mediators. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Examples of cytokines include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma.Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0121] Chemokines are a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus and activation-regulated chemokines (TARC or CCL17).

[0122] "Therapeutic dose," "therapeutic dosage," etc., refer to the amount of cells (such as immune cells or engineered T cells) produced by this method (resulting in a T cell product) that, when used alone or in combination with another therapeutic agent, protect or treat a subject from the onset of disease, or promote disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, and / or prevention of disability or impairment resulting from the onset of the disease. The ability to promote disease regression can be evaluated using various methods known to those skilled in the art, for example, in subjects in clinical trials, in animal model systems predicting efficacy in humans, or by assaying the activity of the drug in in vitro assays. In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (for example, for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject that is not a patient. T cells may be administered in a therapeutic dose. For example, a therapeutic dose of T cells is at least about 10 4 Cells, at least about 10 5 Cells, at least about 10 6 Cells, at least about 10 7 Cells, at least about 108 Cells, at least about 10 9 , or at least about 10 10 It is possible. In another embodiment, the therapeutically effective amount of T cells is about 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of CAR T cells is approximately 2 × 10⁻⁶ 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 Cells / kg, or approximately 9 x 10⁻⁶ 7 The value is cells / kg. In some embodiments, the therapeutically effective dose of CAR-positive surviving T cells is approximately 1 × 10⁶ cells per kg of body weight. 6 ~about 2×10 6 These are CAR-positive viable T cells, and the maximum dose is approximately 1 × 10⁶ 8 These are CAR-positive surviving T cells. In some embodiments, the therapeutically effective dose of CAR-positive surviving T cells is approximately 0.4 × 10⁻⁶ 8 ~about 2×10 8 These are CAR-positive surviving T cells. In some embodiments, the therapeutically effective dose of CAR-positive surviving T cells is approximately 0.4 × 10⁻⁶ 8 , about 0.5×10 8 , about 0.6×10 8 , about 0.7×10 8 , about 0.8×10 8 , about 0.9×10 8, about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , or approximately 2.0 × 10 8 These are CAR-positive surviving T cells.

[0123] As used herein, the term “lymphocyte” may include natural killer (NK) cells, T cells, NK-T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that is a major component of the innate immune system. NK cells reject virus-infected tumors and cells through the process of apoptosis or programmed cell death. They are named “natural killers” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a differentiated organ of the immune system, is primarily responsible for the maturation of T cells.

[0124] There are several types of “immune cells,” which include, but are not limited to, macrophages (e.g., tumor-associated macrophages), neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor-infiltrating lymphocytes (TILs), myeloid-derived suppressor cells (MDSCs), and dendritic cells. The term also includes the precursors of these immune cells. Hematopoietic stem cells and / or progenitor cells may be derived from bone marrow, umbilical cord blood, adult peripheral blood after cytokine recruitment, etc., by methods known in the art. Some progenitor cells may differentiate into lymphoid cells, for example, lymphoid hematopoietic stem cells or progenitor cells. Additional examples of immune cells that may be used in immunotherapy are described in U.S. Patent Application Publication 2018 / 0273601, which is incorporated herein by reference in its entirety.

[0125] Several types of T cells, namely helper T cells (e.g., CD4+ cells, effector T cells) EFF cell), cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell or killer T cell), memory T cell ((i) stem memory T cell) SCM The cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting numerous functional attributes specific to memory cells. (ii) Central Memory T CM The cells express L-selectin and CCR7 + CD45RO + (iii) However, Effector Memory T EM T cells do not express L-selectin or CCR7, but they do express CD45RO and produce effector cytokines such as IFNγ and IL-4. Regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells are also present. T cells found within tumors are called "tumor-infiltrating lymphocytes" (TILs). B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, and the name originates from this.

[0126] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells become CD4 + or CD8 + They appear as either naive T cells. In these naive states, T cells are L-selectin (CD62L +) express IL-7 receptor-α (IL-7R-α) and CD132, but do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" also refers to a T cell that exhibits phenotypic characteristics of either naive T cells or immature T cells, such as T SCM cells or T CM- cells. For example, immature T cells may express one or more of L-selectin (CD62L + ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive or immature T cells can be contrasted with terminally differentiated effector T cells such as T EM cells and T EFF cells.

[0127] As referred to herein, "T cell function" refers to the normal characteristics of healthy T cells. T cell function may include T cell proliferation, T cell activity, and / or cytolytic activity. In one embodiment, the methods of the present application for preparing T cells under specific oxygen and / or pressure conditions increase one or more T cell functions, thereby making the T cells more suitable and / or more potent for therapeutic purposes. In some embodiments, the T cells prepared according to the method have increased T cell function compared to those under conditions lacking specific oxygen and / or pressure. In other embodiments, the T cells prepared according to the method would have increased T cell proliferation compared to T cells cultured under conditions lacking specific oxygen and / or pressure. In additional embodiments, the T cells prepared according to the method have increased T cell activity compared to T cells cultured under conditions lacking specific oxygen and / or pressure. In further embodiments, the T cells prepared according to the method have increased cytolytic activity compared to T cells cultured under conditions lacking specific oxygen and / or pressure.

[0128] The terms "proliferation" and "multiplication" refer to the ability of cells to increase in number through cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation may occur in vitro, for example, during T cell culture, or in vivo, for example, after administration of immunotherapy (e.g., T cell therapy). Cell proliferation may be measured or determined by the methods described herein or by methods known in the art. For example, cell proliferation may be measured or determined by viable cell density (VCD) or total viable cell number (TVC). VCD or TVC may be theoretical (aliquots or samples are taken from the culture at a specific time point, the cell number is determined, and then the cell number is multiplied using the culture volume at the start of the test) or experimental (aliquots or samples are taken from the culture at a specific time point, the cell number is determined, and then the cell number is multiplied using the actual culture volume at that time point). The term "T cell activity" refers to any activity common to healthy T cells. In one embodiment, T cell activity includes cytokine production (e.g., INFγ, IL-2, and / or TNFα). In other embodiments, T cell activity includes the production of interferon-γ (IFNγ or IFN-γ), tissue necrosis factor α (TNFα or IFNα), and one or more cytokines selected from both. Terms such as "cytolytic activity" and "cytotoxicity" refer to the ability of T cells to destroy target cells. In one embodiment, the target cells are cancer cells, e.g., tumor cells. In other embodiments, the T cells express a chimeric antigen receptor (CAR) or T cell receptor (TCR), and the target cells express a target antigen.

[0129] The terms “genetically modified,” “gene-edited,” or “modified” refer to methods of altering a cell’s genome, including, but not limited to, deleting coding regions or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In one embodiment, the modified cells are lymphocytes, such as T cells, which can be obtained from either a patient or a donor. The cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), and incorporated into the cell’s genome.

[0130] The terms “transduction” and “transduced” refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0131] The chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs) of this application are genetically engineered receptors. These engineered receptors can be readily inserted into immune cells, such as T cells, and expressed by immune cells according to techniques known in the art. In CARs, a single receptor can recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells that possess or express that antigen. If these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells. In one embodiment, cells prepared according to this application are cells having a chimeric antigen receptor (CAR) or T cell receptor, comprising an antigen-binding molecule, a costimulatory domain, and an activating domain. The costimulatory domain may include an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain includes a hinged or cleaved hinged domain.

[0132] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Ab, cytokines, and complement) produced by either these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of a vertebrate of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0133] The term "immunotherapy" refers to the treatment of a person who is suffering from a disease or at risk of suffering from or relapsing from a disease, by means of methods including inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T-cell and NK-cell therapies. T-cell therapies may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy, and allogeneic T-cell transplantation. Those skilled in the art will understand that the methods of preparing immune cells disclosed herein enhance the efficacy of any cancer or transplanted T-cell therapy. Examples of T-cell therapy are described in U.S. Patent Publications 2014 / 0154228 and 2002 / 0006409; U.S. Patents 7,741,465; U.S. Patents 6,319,494; and U.S. Patents 5,728,388; and PCT Publication WO2008 / 081035 (these are incorporated in their entirety by reference).

[0134] The term "engineered autologous cell therapy," also known as adoptive cell transfer and sometimes abbreviated as "eACT(trademark)," refers to the process of harvesting a patient's own T cells and subsequently genetically modifying them to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells can be engineered, for example, to express a chimeric antigen receptor (CAR) or T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-strand variable fragment (scFv) specific to a particular tumor antigen, linked to an intracellular signaling region containing a costimulatory domain and an activation domain.The co-stimulatory domains include, for example, CD28, CTLA4, CD16, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), inducible T cell costimulator (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICA M-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp 46, CD19, CD4, CD8, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domain may be derived from ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof. The activation domain may be derived from CD3, such as CD3ζ, ε, δ, or γ.In one embodiment, the CAR is designed to have two, three, four, or more co-stimulatory domains. The CAR scFv can be designed to target a transmembrane protein expressed by cells of B cell lineages, including, but not limited to, NHL, CLL, and non-T cell ALL, including all normal B cells and B cell malignancies, such as CD19. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Applications Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated in whole by reference.

[0135] As used herein, “co-stimulatory signal” refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, elicits an upregulation or downregulation of a T cell response, such as, but not limited to, proliferation and / or major molecules.

[0136] As used herein, “costimulatory ligand” encompasses molecules on antigen-presenting cells that specifically bind to homologous costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses, such as proliferation, activation, and differentiation, but is not limited to these. In addition to the primary signals provided by stimulatory molecules, costimulatory ligands induce signals, for example, by binding the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Examples of co-stimulatory ligands, though not limited to them, include 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin β receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD)L1. Examples of co-stimulatory ligands, though not limited to them, include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0137] A "costimulatory molecule" is a congenital junction partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells, such as proliferation, but is not limited to this.Co-stimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD33, and CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CDl Examples include la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules, SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleaved forms, or combinations thereof.

[0138] In some embodiments, the cells of this application can be obtained via T cells obtained from a subject. In one embodiment, T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a suitable buffer or medium for further processing. In some embodiments, cells are washed with any solution (e.g., a solution with a neutral pH or PBS) or culture medium. As understood, the washing step may be performed using, for example, a semi-automated flow-through centrifuge, e.g., a Cobe® 2991 cell processor, a Baxter CytoMate®, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or in other salines, with or without buffers. In some embodiments, undesirable components are removed from the apheresis sample. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0139] In some embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes, for example, using centrifugation with a PERCOLL® gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the Art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. In some embodiments, cell sorting and / or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate the desired cell population for use in the present disclosure.

[0140] In one embodiment, CD3+ T cells are isolated from PBMCs using Dynabeads coated with an anti-CD3 antibody. CD8+ and CD4+ T cells are further isolated separately by positive selection using CD8 microbeads (e.g., Miltenyi Biotec) or CD4 microbeads (e.g., Miltenyi Biotec).

[0141] In some embodiments, PBMCs are used directly for genetic modification (such as CAR) of immune cells using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated, and both cytotoxic T lymphocytes and helper T lymphocytes are sorted into subpopulations of naive T cells, memory T cells, and effector T cells before or after genetic modification and / or proliferation.

[0142] One or more immune cells described herein can be obtained from any source, including, for example, a human donor. The donor may be a subject (i.e., an autologous donor) that requires treatment with an immune cell population produced by the method herein, or an individual (i.e., an allogeneic donor) that provides a lymphocyte sample to be used to treat another individual or cancer patient at the time of generating the cell population produced by the method herein. Immune cells can be differentiated in vitro from a hematopoietic stem cell population, or immune cells can be obtained from a donor. A population of immune cells can be obtained from a donor by any suitable method used in the art. For example, a population of lymphocytes can be obtained by any suitable extracorporeal method, venipuncture, or other blood collection method that yields a blood sample in which lymphocytes may or may not be present. A population of lymphocytes can be obtained by apheresis. One or more immune cells can be collected from any tissue containing one or more immune cells, including but not limited to tumors. A tumor or a portion thereof is collected from a subject, and one or more immune cells are isolated from the tumor tissue. In the methods disclosed herein, any T cells, including any immune cells suitable for T cell therapy, can be used. For example, one or more cells useful for this application may be selected from the group consisting of tumor-infiltrating lymphocytes (TILs), cytotoxic T cells, CAR T cells, engineered TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In addition, T cells may be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from subjects using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. T cells can also be obtained from artificial thymic organoid (ATO) cell culture systems, in which the human thymic environment is replicated to support the efficient ex vivo differentiation of primary and reprogrammed pluripotent stem cell-derived T cells.Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, International Publication No. 2015 / 120096, and International Publication No. 2017 / 070395, all of which are incorporated herein by reference in their entirety for the purpose of illustrating these methods. In one embodiment, the T cells are tumor-infiltrating leukocytes. In a particular embodiment, one or more T cells express CD8, for example, CD8. + These are T cells. In other embodiments, one or more T cells express CD4, for example, CD4 + These are T cells. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, International Publication No. 2015 / 120096, and International Publication No. 2017 / 070395, all of which are incorporated herein by reference in their entirety for the purpose of illustrating these methods.

[0143] Immune cells and their progenitor cells can be isolated by available methods (see, for example, Rowland-Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York (1999)). Sources of immune cells or their progenitor cells include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow, or other hematopoietic cell sources. Negative selection methods can be used to remove cells that are not the desired immune cells. Furthermore, positive selection methods can isolate or concentrate the desired immune cells or their progenitor cells, or a combination of positive and negative selection methods may be used. Monoclonal antibodies (MAbs) are useful in both positive and negative selection to identify markers associated with specific cell lineages and / or differentiation stages. When isolating a specific type of cell, such as a specific type of T cell, various cell surface markers or combinations of markers may be used to isolate the cells, as is well known in the art, including but not limited to CD3, CD4, CD8, and CD34 (for hematopoietic stem cells and progenitor cells) (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa NJ 2000; De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009)).

[0144] PBMCs can be used directly for genetic modification by immune cells (such as CARs). After isolating PBMCs, T lymphocytes are further isolated, and both cytotoxic T lymphocytes and helper T lymphocytes are sorted into subpopulations of naive T cells, memory T cells, and effector T cells before or after genetic modification and / or proliferation. In one embodiment, CD8+ cells may be further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In other embodiments, the expression of phenotypic markers for central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In certain embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In additional embodiments, CD4+ T cells may be further classified into subpopulations. For example, CD4+ T helper cells may be sorted into naive cells, central memory cells, and effector cells by identifying cell populations that possess cell surface antigens.

[0145] The methods described herein further include enriching or preparing a population of immune cells obtained from a donor, from the time of collection from the donor until exposure to one or more cells obtained from the donor subject. The enrichment of immune cells, for example, a population of one or more T cells, may be achieved by separation medium (e.g., FICOLL-PAQUE®, ROSETTESEP® HLA Total Lymphocyte Enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog number 0850494X), etc.), cell size, shape or density separation by filtration or elution, immunomagnetic separation (e.g., magnetically activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation.

[0146] In one embodiment, the T cells are obtained from a donor subject. In other embodiments, the donor subject is a human patient suffering from cancer or a tumor. In additional embodiments, the donor subject is a human patient not suffering from cancer or a tumor. The present application also provides a composition or formulation comprising a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the composition or formulation comprises an excipient. The terms "crude product" and "formulation" are used interchangeably herein. The terms composition, therapeutic composition, therapeutically effective composition, pharmaceutical composition, pharmaceutically effective composition, and pharmaceutically acceptable composition are used interchangeably herein. The composition can be selected for parenteral delivery, inhalation, or delivery via the gastrointestinal tract such as oral. The composition can be prepared by methods known to those skilled in the art. A buffer is used to maintain the composition at physiological pH or slightly lower pH, typically within a pH range of about 5 to about 8. When parenteral administration is contemplated, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution comprising the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. By way of example, the parenteral injection vehicle is sterile distilled water in which the composition described herein is formulated as a properly preserved sterile isotonic solution, with or without at least one additional therapeutic agent. Preparation involves formulating the desired agent using beads or liposomes which are polymeric compounds (such as polylactic acid or polyglycolic acid) that allow for controlled or sustained release of the product, which are then delivered by depot injection. Further, an implantable drug delivery device can be used to introduce the desired therapeutic agent.

[0147] In some embodiments, the donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, the donor T cells for use in T cell therapy are obtained from a subject other than the patient. The T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells is at least about 10 4 cells, at least about 10 5Cells, at least about 10 6 Cells, at least about 10 7 Cells, at least about 10 8 Cells, at least about 10 9 , or at least about 10 10 It is possible. In another embodiment, the therapeutically effective amount of T cells is about 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of CAR T cells is approximately 2 × 10⁻⁶ 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 Cells / kg, or approximately 9 x 10⁻⁶ 7 The value is cells / kg. In some embodiments, the therapeutically effective dose of CAR-positive surviving T cells is approximately 1 × 10⁶ cells per kg of body weight. 6 ~about 2×10 6 These are CAR-positive viable T cells, and the maximum dose is approximately 1 × 10⁶ 8 These are CAR-positive surviving T cells.

[0148] As used herein, “patient” includes any human being suffering from a disease or disorder such as cancer (e.g., lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably herein. The term “donor subject” herein refers to a subject from which cells are collected for further in vitro manipulation. A donor subject may be a cancer patient to be treated with the cell population produced by the method herein (i.e., an autologous donor), or an individual to provide a lymphocyte sample to be used to treat another individual or cancer patient at the time of production of the cell population produced by the method herein (i.e., an allogeneic donor). A subject to receive cells prepared by this method may be referred to as a “recipient subject.”

[0149] The terms "stimulation" and "to stimulate" refer to the primary response induced by the binding of a stimulating molecule to its homologous ligand, and this binding mediates a signaling event. A "stimulating molecule" is a molecule on a T cell, such as the T cell receptor (TCR) / CD3 complex, that specifically binds to homologous stimulating ligands present on antigen-presenting cells. A "stimulating ligand," when present on antigen-presenting cells (e.g., artificial antigen-presenting cells (aAPCs), dendritic cells, B cells, etc.), specifically binds to stimulating molecules on T cells, thereby mediating primary T cell responses, including, but not limited to, activation, initiation of an immune response, and proliferation. Examples of stimulating ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. "Activated" or "active" as used herein refers to a stimulated T cell. Activated T cells can be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

[0150] The term “exogenous activator” refers to any activator derived from an external source. For example, exogenous anti-CD3 antibody, anti-CD28 antibody, IL-2, exogenous IL-7, or exogenous IL-15 may be commercially available or produced by recombinant. When added to or in contact with one or more T cells, “exogenous IL-2,” “exogenous IL-7,” or “exogenous IL-15” indicates that such IL-2, IL-7, and / or IL-15 are not produced by the T cells. T cells before being mixed with “exogenous” IL-2, IL-7, or IL-15 may contain trace amounts produced by the T cells or isolated from subjects containing T cells (i.e., endogenous “exogenous” IL-2, IL-7, or IL-15). One or more T cells described herein can be contacted with exogenous anti-CD3 antibody, anti-CD28 antibody, "exogenous" IL-2, IL-7 and / or IL-15 by any means known in the art, including the addition of isolated "exogenous" IL-2, IL-7 and / or IL-15 to a culture, the addition of anti-CD3 antibody, anti-CD28 antibody, "exogenous" IL-2, IL-7 and / or IL-15 to a culture medium, or the expression of "exogenous" IL-2, IL-7 and / or IL-15 by one or more cells in a culture other than one or more T cells, such as by a feeder layer.

[0151] As used herein, the term “in vitro cells” refers to any cells cultured ex vivo. In one embodiment, T cells are given as in vitro cells.

[0152] The term "persistence" refers, for example, to the ability to maintain one or more transplanted immune cells, or their offspring (e.g., differentiated or matured T cells), administered to a subject at a detectable level within the subject for a certain period of time. As used herein, increasing the persistence of one or more transplanted immune cells, or their offspring (e.g., differentiated or matured T cells), means extending the period during which the transplanted immune cells are detectable in the subject after administration. For example, the in vivo persistence of one or more transplanted immune cells may be extended by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. In addition, the in vivo persistence of one or more transplanted immune cells may be extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to one or more transplanted immune cells not prepared by the methods disclosed herein.

[0153] The terms “reduce” and “decrease” are used interchangeably herein and refer to any change smaller than the original. “Reduce” and “decrease” are relative terms and require a comparison of before and after measurements. “Reduce” and “decrease” include complete depletion. The term “regulate” T cell maturation, when used herein, refers to the use of any intervention described herein to control the maturation and / or differentiation of one or more cells, such as T cells. For example, regulation refers to the inactivation, delay, or inhibition of T cell maturation. In another example, regulation refers to the acceleration or promotion of T cell maturation. The term “delay or inhibition of T cell maturation” refers to keeping one or more T cells in an immature or undifferentiated state. For example, “delay or inhibition of T cell maturation” refers to keeping T cells naive or T CM It can refer to maintaining a state, T EM or T EFF This is in contrast to progressing to a certain state. In addition, "delay or inhibition of T cell maturation" refers to immature or undifferentiated T cells (e.g., naive T cells and / or T cells) within a mixed population of T cells. CM This can refer to increasing or enriching the overall percentage of T cells. The state of T cells (e.g., mature or immature) can be determined, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of T cells. For example, the presence of one or more markers selected from the group consisting of L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof may indicate a less mature, undifferentiated T cell.

[0154] "Treatment" or "treating" a subject / patient means any type of intervention or process performed on the subject / patient, or administration to the subject / patient, for the purpose of restoring, alleviating, improving, inhibiting, slowing or preventing the onset, exacerbation, development, severity or relapse of symptoms, complications or conditions, or biochemical signs associated with the disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0155] Various aspects of this application are described in more detail in the following subsections.

[0156] Patients with B-cell malignancies exhibiting high levels of circulating CD19-expressing tumor cells represent a population with very high unmet needs. For example, mantle cell lymphoma (MCL) is difficult to treat in its relapsed or refractory state and remains untreatable. There is no standard of care for second-line and more advanced chemotherapy. Treatment options include cytotoxic chemotherapy, proteasome inhibitors, immunomodulators, tyrosine kinase inhibitors, and stem cell transplantation (autologous [ASCT] and allogeneic stem cell transplantation [allo-SCT]). The choice of regimen is influenced by prior treatment, comorbidities, and the tumor's sensitivity to chemotherapy agents. Despite the high initial response rates observed with Bruton's tyrosine kinase inhibitors (BTK inhibitors), most patients ultimately develop progressive disease. Novel treatment strategies are needed to improve the poor prognosis of patients with r / r MCL that is not effectively controlled by chemoimmunotherapy, stem cell transplantation, and BTK inhibitors.

[0157] Products used in anti-CD19 CAR T cell therapy or CD19 CAR-T can be produced from the patient's own T cells via leukocyte apheresis suitable for B-cell malignancies with a circulating tumor cell volume, in order to minimize CD19-expressing tumor cells in the final product. Leukocyte-derived T cells collected from the leukocyte apheresis product can be enriched by selection of CD4+ / CD8+ T cells, activated with anti-CD3 and anti-CD28 antibodies, and / or transduced with a viral vector containing the anti-CD19 CAR gene. Further details of this method can be found in international applications PCT / US2015 / 014520, published as international publication 2015 / 120096, and international applications PCT / US2016 / 057983, published as international publication 2017 / 070395. In one embodiment, the cells are not treated with AKT inhibitors, IL-7, and IL-15. These manipulated T cells can be proliferated to generate a sufficient number of cells to achieve a therapeutic effect. Such a process can eliminate malignant and normal B cells expressing CD19, thereby reducing the activation, proliferation, and depletion of anti-CD19 CAR T cells.

[0158] The activation, transduction, and / or proliferation of immune cells may be carried out for any preferred period of time that allows for the production of (i) a sufficient number of cells in a population of engineered immune cells for at least one dose to be administered to a patient, (ii) a population of engineered immune cells having a preferred proportion of immature cells compared to a typical longer process, or (iii) a preferred period of time that allows for the production of both (i) and (ii). The preferred period may be determined by several parameters, including a population of one or more cells, cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and / or other variables. The activation period may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more than 21 days. The activation period by the method of this application is shorter compared to proliferation methods known in the art. For example, the activation period may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 75% shorter. Furthermore, the growth period may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more than 21 days. The growth period by the method of this application is reduced compared to growth methods known in the art. For example, the proliferation period may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 75% shorter. In one embodiment, the cell proliferation period is about 3 days, and the period from enrichment of the cell population to the generation of manipulated immune cells is about 6 days.

[0159] Delays or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, delays or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more biomarkers. The presence of one or more biomarkers can be detected by any method known in the art, including but not limited to immunohistochemistry and / or fluorescence-activated cell sorting (FACS). One or more biomarkers may be L-selectin (CD62L + ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof are selected from the group. In certain embodiments, delay or inhibition of maturation or differentiation of one or more T cells or DC cells is performed by L-selectin (CD62L + This can be measured by detecting the presence of one or more of ), IL-7Rα, and CD132. Those skilled in the art will recognize that this method may increase the relative proportion of immature and undifferentiated T cells or DC cells in the collected cell population, but some mature and differentiated cells may still be present. As a result, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells may be measured by calculating the total percentage of immature and undifferentiated cells in the cell population before and after exposure of one or more cells obtained from the subject to hypoxic culture conditions with or without pressure above atmospheric pressure. The method disclosed herein may increase the percentage of immature and undifferentiated T cells in a T cell population.

[0160] The methods described herein further include stimulating a population of cells, such as lymphocytes, with one or more T cell stimulants to generate a population of activated T cells under appropriate conditions. A population of activated T cells can be generated using any combination of one or more suitable T cell stimulants, including but not limited to antibodies or functional fragments thereof that target T cell stimulating molecules or co-stimulating molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies (such as OKT-3), anti-CD28 antibodies, or functional fragments thereof), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), porkweed mitogen (PWM)), or natural ligands for T cell stimulating molecules or co-stimulating molecules.

[0161] Preferred conditions for stimulating or activating the immune cell populations described herein further include a temperature for a certain period of time and / or in the presence of a certain level of CO2. The temperature for stimulation may be about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34-38°C, about 35-37°C, about 36-38°C, about 36-37°C, or about 37°C.

[0162] Another condition for stimulating or activating the immune cell population described herein may further include the duration of stimulation or activation. The duration of stimulation is approximately 24–72 hours, approximately 24–36 hours, approximately 30–42 hours, approximately 36–48 hours, approximately 40–52 hours, approximately 42–54 hours, approximately 44–56 hours, approximately 46–58 hours, approximately 48–60 hours, approximately 54–66 hours, approximately 60–72 hours, approximately 44–52 hours, approximately 40–44 hours, approximately 40–48 hours, approximately 40–52 hours, or approximately 40–56 hours. In one embodiment, the duration of stimulation is approximately 48 hours or at least approximately 48 hours.

[0163] Other conditions for stimulating or activating the immune cell populations described herein may further include CO2 levels. CO2 levels for stimulation are about 1.0–10% CO2, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2, about 3–7% CO2, about 4–6% CO2, or about 4.5–5.5% CO2. In one embodiment, the CO2 level for stimulation is about 5% CO2.

[0164] Conditions for stimulating or activating an immune cell population may further include temperature, a certain period of time for stimulation, and / or the presence of a certain level of CO2, in any combination thereof. For example, the step of stimulating an immune cell population may include stimulating the immune cell population with one or more immune cell stimulants at a temperature of about 36–38°C for about 44–52 hours and in the presence of a CO2 level of about 4.5–5.5% CO2. One or more immune cells of this application may be administered to a subject for use in immunotherapy or cell therapy. Therefore, one or more immune cells may be collected from a subject requiring immunotherapy or cell therapy. Once collected, one or more immune cells may be processed for any preferred period of time before being administered to a subject.

[0165] The concentration, quantity, or population of lymphocytes or resulting products produced by the method described herein is approximately 1.0 to 10.0 × 10⁶. 6 The concentration is cells / mL. In certain embodiments, the concentration is approximately 1.0–2.0 × 10⁻⁶. 6 cells / mL, approximately 1.0~3.0×10 6 cells / mL, approximately 1.0~4.0×10 6 cells / mL, approximately 1.0~5.0×10 6 cells / mL, approximately 1.0~6.0×10 6 cells / mL, approximately 1.0~7.0×10 6 cells / mL, approximately 1.0~8.0×10 6 cells / mL, 1.0~9.0×10 6 cells / mL, approximately 1.0~10.0×10 6 cells / mL, approximately 1.0~1.2×10 6 cells / mL, approximately 1.0~1.4×106 cells / mL, approximately 1.0~1.6×10 6 cells / mL, approximately 1.0~1.8×10 6 cells / mL, approximately 1.0~2.0×10 6 Cells / mL, at least about 1.0 × 10⁶ 6 Cells / mL, at least about 1.1 × 10⁶ 6 Cells / mL, at least approximately 1.2 × 10⁶ 6 Cells / mL, at least approximately 1.3 × 10⁶ 6 Cells / mL, at least approximately 1.4 × 10⁶ 6 Cells / mL, at least approximately 1.5 × 10⁶ 6 Cells / mL, at least approximately 1.6 × 10⁶ 6 Cells / mL, at least approximately 1.7 × 10⁶ 6 Cells / mL, at least approximately 1.8 × 10⁶ 6 Cells / mL, at least approximately 1.9 × 10⁶ 6 Cells / mL, at least approximately 2.0 × 10⁶ 6 Cells / mL, at least approximately 4.0 × 10⁶ 6 Cells / mL, at least approximately 6.0 × 10⁶ 6 Cells / mL, at least approximately 8.0 × 10⁶ 6 cells / mL, or at least about 10.0 × 10⁴ 6 The concentration is cells / mL.

[0166] Anti-CD3 antibodies (or their functional fragments), anti-CD28 antibodies (or their functional fragments), or combinations of anti-CD3 and anti-CD28 antibodies may be used in conjunction with, or independently of, exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without atmospheric pressure, in order to stimulate a population of lymphocytes. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibodies or their functional fragments (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)) may be used. In some embodiments, antibodies may be commercially available from distributors known in the art, including but not limited to Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 Pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Furthermore, those skilled in the art will understand methods for producing anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, one or more T-cell stimulants used in a step of stimulating a population of lymphocytes include antibodies or functional fragments thereof that target T-cell stimulating or co-stimulating molecules in the presence of T-cell cytokines. In one embodiment, one or more T-cell stimulants include an anti-CD3 antibody and IL-2. In a particular embodiment, the T-cell stimulant includes an anti-CD3 antibody at a concentration of 50 ng / mL. The concentration of anti-CD3 antibody is approximately 20 ng / mL to 100 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL. In alternative embodiments, T cell activation is not required.

[0167] The method described herein further comprises transducing a population of activated immune cells with a viral vector containing a nucleic acid molecule encoding a cell surface receptor using one or more viral transduction cycles to produce a population of transduced immune cells. Several recombinant viruses are used as viral vectors to deliver genetic material to cells. Viral vectors that may be used according to the transduction step include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenovirus vectors, and recombinant adeno-associated viral (AAV) vectors, and may be any ecotropic or amphotropic viral vector. The method further comprises transducing one or more immune cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated immune cells is the MSGV1γ retroviral vector. In one embodiment, the viral vector used to transduce a population of activated immune cells is the PG13-CD19-H3 vector described in Kochenderfer, J.Immunother.32(7):689-702(2009). According to one embodiment of this specification, the viral vector is grown in suspension culture in a medium specific to viral vector production, referred herein as viral vector inoculum. Any suitable growth medium and / or supplement for growing the viral vector may be used with the viral vector inoculum according to the method described herein. According to some embodiments, the viral vector inoculum is then added to a serum-free culture medium described below during the transduction step. In some embodiments, one or more immune cells may be transduced with a retrovirus. In one embodiment, the retrovirus comprises a heterologous gene encoding a cell surface receptor. In another embodiment, the cell surface receptor may bind to an antigen on the surface of a target cell, for example, on the surface of a tumor cell.In addition to optionally exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without pressure exceeding atmospheric pressure, the conditions for transducing a population of activated immune cells described herein may include a specific temperature and / or a specific level of CO2 for a specific time. The temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34-38°C, about 35-37°C, about 36-38°C, or about 36-37°C. In one embodiment, the temperature for transduction is about 37°C. A predetermined temperature for transduction may be about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, or about 39°C, about 34-39°C, or about 35-37°C. In one embodiment, a predetermined temperature for transduction may be about 36-38°C, about 36-37°C, or about 37°C. The time for transduction is approximately 12–36 hours, approximately 12–16 hours, approximately 12–20 hours, approximately 12–24 hours, approximately 12–28 hours, approximately 12–32 hours, approximately 20 hours, or at least approximately 20 hours, approximately 16–24 hours, approximately 14 hours, at least approximately 16 hours, at least approximately 18 hours, at least approximately 20 hours, at least approximately 22 hours, at least approximately 24 hours, or at least approximately 26 hours. The CO2 level for transduction is approximately 1.0–10% CO2, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0%, approximately 6.0%, approximately 7.0%, approximately 8.0%, approximately 9.0%, approximately 10.0% CO2, approximately 3–7% CO2, approximately 4–6% CO2, approximately 4.5–5.5% CO2, or approximately 5% CO2.

[0168] The introduction of the activated immune cell population described herein may be carried out in any combination of a specific period, a specific temperature, and / or the presence of a specific level of CO2, such as a temperature of about 36–38°C, about 16–24 hours, and a CO2 level of about 4.5–5.5% CO2. The immune cells may be prepared by a combination of any one of the methods of this application and any method of preparing T cells for immunotherapy, any such method of preparation is incorporated herein by reference in whole by international publications 2015 / 120096 and 2017 / 070395 of the PCT Publications, which are incorporated herein by whole for the purpose of illustrating these methods; any method used for the preparation of Tisagenlecleucel / Kymriah®; any method used for the preparation of “ready-made” T cells for immunotherapy; and any other method of preparing lymphocytes for administration to humans. The preparation process may be adapted to remove circulating tumor cells from cells obtained from a patient.

[0169] CAR-T cells can be engineered to express other molecules and may be any one of the following: first, second, third, fourth, fifth or more CAR-T cells; arm-type CAR-T cells, motile CAR-T cells, TRUCK T cells, switch receptor CAR-T cells; gene-edited CAR-T cells; dual receptor CAR-T cells; suicide CAR-T cells, drug-inducible CAR-T cells, synNotch-inducible CAR-T cells; and inhibitory CAR-T cells, or any other exemplary types available in the art. In one embodiment, the T cells are autologous T cells. In one embodiment, the T cells are autologous stem cells (for autologous stem cell therapy or ASCT). In one embodiment, the T cells are non-autologous T cells.

[0170] Cells (such as immune cells or T cells) are genetically modified after isolation or selection using known methods, or are activated and / or proliferated in vitro (or differentiated in the case of progenitor cells) before genetic modification. Immune cells, e.g., T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), activated in vitro, and / or proliferated. Methods for activating and proliferating T cells can be found in U.S. Patents 6,905,874, 6,867,041, and 6,797,514, and International Publication 2012 / 079000 of the PCT Publications, which are incorporated herein by reference in their entirety. Generally, such methods may involve contacting PBMCs or isolated T cells with stimulants and co-stimulants that can be bound to beads or other surfaces, e.g., anti-CD3 and / or anti-CD28 antibodies, in a culture medium containing specific cytokines such as IL-2. The Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells, may be used. T cells can be activated and stimulated and proliferated with suitable supporting cells, antibodies, and / or cytokines, as described in U.S. Patent Nos. 6,040,177 and 5,827,642, and International Publication No. 2012 / 129514 of the PCT Publications, the entire contents of which are incorporated herein by reference.

[0171] The cell surface receptor expressed by the engineered immune cells may be any antigen or molecule targeted by the CAR, e.g., anti-CD19 CAR, FMC63-28Z CAR, or FMC63-CD828BBZ CAR (Kochenderfer et al., J Immunother. 2009, 32(7):689; Locke et al., Blood 2010, 116(20):4099), both of which are incorporated herein by reference. In certain embodiments, a given dose of engineered immune cells may be more than about 1 million and less than about 3 million transduced T cells / kg. In one embodiment, a given dose of engineered T cells may be about 1 million to about 2 million transduced T cells (cells / kg) per kilogram of body weight. A given dose of engineered T cells may be 1 million to about 2 million, at least about 2 million to about 3 million per kilogram of body weight. The number of transduced T cells (cells / kg) may be less than 1 million. In one embodiment, a given dose of engineered T cells may be about 2 million transduced T cells / kg. In another embodiment, a given dose of engineered T cells may be at least about 2 million transduced T cells / kg. Examples of given doses of engineered T cells may be about 2.0 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced modified T cells / kg.

[0172] The methods described herein involve increasing or concentrating a population of one or more transduced immune cells for a certain period of time to produce a population of engineered immune cells. The period for proliferation may be (i) a sufficient number of cells in the population of engineered immune cells for at least one dose to be administered to a patient, (ii) a population of engineered immune cells having a preferred proportion of immature cells compared to a typical longer process, or (iii) any preferred period that allows for the production of both (i) and (ii). This period depends on the cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and other variables. A predetermined period for proliferation may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more than 21 days. In one embodiment, the period for proliferation of the present method is shorter than those known in the art. For example, the predetermined proliferation period may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 75% shorter. In one example, the proliferation period is approximately 3 days, and the period from the enrichment of the lymphocyte population to the generation of manipulated immune cells is approximately 6 days.

[0173] Conditions for growing a population of transduced immune cells may include temperature and / or the presence of a certain level of CO2. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 35-37°C, about 36-37°C, or about 37°C. The level of CO2 is 1.0-10% CO2, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% CO2, about 4.5-5.5% CO2, about 5% CO2, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2.

[0174] Each step of the method described herein may be performed in a closed system. The closed system may be a closed-bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife Cell Culture bags). The cell culture bags used in the closed-bag culture system may be coated with recombinant human fibronectin fragments during the transduction step. The recombinant human fibronectin fragments may comprise three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. By using recombinant human fibronectin fragments, the gene efficiency of retroviral transduction of immune cells can be increased by assisting the co-localization of target cells and viral vectors. In one embodiment, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). Cell culture bags contain approximately 1-60 μg / mL, or approximately 1-40 μg / mL, approximately 1-20 μg / mL, 20-40 μg / mL, 40-60 μg / mL, approximately 1 μg / mL, approximately 2 μg / mL, approximately 3 μg / mL, approximately 4 μg / mL, approximately 5 μg / mL, approximately 6 μg / mL, approximately 7 μg / mL, approximately 8 μg / mL, approximately 9 μg / mL, approximately 10 μg / mL, approximately 11 μg / mL, approximately 12 μg / mL, approximately 13 μg / mL, approximately 14 μg / mL, approximately 15 μg / mL, approximately 16 μg / mL, approximately 17 μg / mL, approximately 18 μg / mL, approximately 19 μg / mL, approximately 20 μg / mL, approximately 2-5 μg / mL, approximately 2-10 μg / mL, and approximately 2-20 μg Coated with recombinant human fibronectin fragments at concentrations of approximately 2-25 μg / mL, 2-30 μg / mL, 2-35 μg / mL, 2-40 μg / mL, 2-50 μg / mL, 2-60 μg / mL, at least approximately 2 μg / mL, at least approximately 5 μg / mL, at least approximately 10 μg / mL, at least approximately 15 μg / mL, at least approximately 20 μg / mL, at least approximately 25 μg / mL, at least approximately 30 μg / mL, at least approximately 40 μg / mL, at least approximately 50 μg / mL, or at least approximately 60 μg / mL.In one embodiment, the cell culture bag is coated with at least about 10 μg / mL of recombinant human fibronectin fragment. The cell culture bag used in the closed-bag culture system may be optionally blocked with human albumin serum (HSA) during the transduction step. In another embodiment, the cell culture bag is not blocked with HSA during the transduction step.

[0175] Populations of engineered immune cells produced by the above method may be optionally cryopreserved for later use. Methods for cryopreserving populations of engineered immune cells are also provided herein. Such methods may include the step of washing and concentrating the population of engineered immune cells with a diluent. For example, the diluent may be physiological saline, 0.9% physiological saline, PlasmaLyte A (PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. HSA may also be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another embodiment, the washing solution is physiological saline, and HSA (5%) is added to the washed and concentrated cells. This method may also include the step of producing a cryopreservation mixture, in which the cryopreservation mixture includes the population of diluted cells in the diluent and a suitable cryopreservation solution. The cryopreservation solution may be any suitable solution, including but not limited to CryoStor10 (BioLife Solutions), and is mixed with the diluent of manipulated immune cells in a 1:1 or 2:1 ratio. HSA may be added to the cryopreserved mixture to provide final concentrations of approximately 1.0–10%, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0%, approximately 6.0%, approximately 7.0%, approximately 8.0%, approximately 9.0%, approximately 10.0%, approximately 1–3% HSA, approximately 1–4% HSA, approximately 1–5% HSA, approximately 1–7% HSA, approximately 2–4% HSA, approximately 2–5% HSA, approximately 2–6% HSA, approximately 2–7% HSA, or approximately 2.5% HSA. Cryopreservation of a population of manipulated immune cells may include washing the cells with 0.9% saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStor® CS10 (final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method also includes a step of freezing the cryopreservation mixture. The cryopreservation mixture contains approximately 1 × 10⁶ of HSA. 6 ~Approx. 1.5×10 7At a cell concentration of cells / mL, the mixture is frozen in a controlled-rate freezer using a defined freeze cycle. This method may also include the step of storing the cryopreservation mixture in gas-phase liquid nitrogen.

[0176] Populations of engineered immune cells produced by the methods described herein may be cryopreserved in predetermined doses. The predetermined dose may be a therapeutically effective dose, which may be any therapeutically effective dose provided below. The predetermined dose of engineered immune cells may depend on the cell surface receptors expressed by the immune cells (e.g., affinity and density of cell surface receptors expressed on the cells), the type of target cell, the nature of the disease or condition being treated, or a combination of both.

[0177] In one embodiment, a population of manipulated T cells can be cryopreserved at a predetermined dose of approximately 1 million manipulated T cells per kilogram of body weight (cells / kg). In a particular embodiment, a population of manipulated T cells can be cryopreserved at a predetermined dose of approximately 500,000 to approximately 1 million manipulated T cells / kg. In a particular embodiment, a population of manipulated T cells can be cryopreserved at a predetermined dose of at least approximately 1 million, at least approximately 2 million, at least approximately 3 million, at least approximately 4 million, at least approximately 5 million, at least approximately 6 million, at least approximately 7 million, at least approximately 8 million, at least approximately 9 million, and at least approximately 10 million manipulated T cells / kg. In other embodiments, the manipulated T cell population can be cryopreserved at predetermined doses of less than 1 million cells / kg, 1 million cells / kg, 2 million cells / kg, 3 million cells / kg, 4 million cells / kg, 5 million cells / kg, 6 million cells / kg, 7 million cells / kg, 8 million cells / kg, 9 million cells / kg, 10 million cells / kg, more than 10 million cells / kg, more than 20 million cells / kg, more than 30 million cells / kg, more than 40 million cells / kg, more than 50 million cells / kg, more than 60 million cells / kg, more than 70 million cells / kg, more than 80 million cells / kg, more than 90 million cells / kg, or more than 100 million cells / kg. In specific embodiments, the manipulated T cell population can be cryopreserved at predetermined doses of approximately 1 million to approximately 2 million manipulated T cells / kg. The manipulated T cell population can be cryopreserved in predetermined volumes of approximately 1 million to 2 million cells / kg, 1 million to 3 million cells / kg, 1 million to 4 million cells / kg, 1 million to 5 million cells / kg, 1 million to 6 million cells / kg, 1 million to 7 million cells / kg, 1 million to 8 million cells / kg, 1 million to 9 million cells / kg, and 1 million to 10 million cells / kg. The predetermined dose of the manipulated T cell population can be calculated based on the subject's body weight. In one example, the manipulated T cell population can be cryopreserved in approximately 0.5 to 200 mL of cryopreservation medium.Furthermore, the manipulated T cell population may be cryopreserved in approximately 0.5 mL, 1.0 mL, 5.0 mL, 10.0 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or approximately 100 mL, 10-30 mL, 10-50 mL, 10-70 mL, 10-90 mL, 50-70 mL, 50-90 mL, 50-110 mL, 50-150 mL, or approximately 100-200 mL of cryopreservation medium. In certain embodiments, the manipulated T cell population may preferably be cryopreserved in approximately 50-70 mL of cryopreservation medium.

[0178] In one embodiment, at least one of the following is carried out using a serum-free culture medium that does not contain added serum: (a) exposing a population of immune cells to exogenous IL-2, exogenous IL-7, exogenous IL-15, and / or other cytokines; (b) stimulating a population of immune cells; (c) transducing an activated population of immune cells; and (d) growing a transduced population of immune cells. In some embodiments, each of (a) to (d) is carried out using a serum-free culture medium that does not contain added serum. As used herein, the terms “serum-free medium” or “serum-free culture medium” mean that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, serum is not added to the medium as a separate and distinct component for the purpose of supporting the viability, activation, and growth of cultured cells. Any suitable immunocell growth medium can be used to culture cells in suspension according to the method described herein. For example, an immunotherapy medium may include, but is not limited to, a sterile low-glucose solution containing suitable amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OPTMIZER® (Life Technologies). In contrast to typical methods for generating engineered immune cells, the methods described herein can use culture media that are not supplemented with serum (e.g., human or bovine).

[0179] This application provides various methods for treating cancer using T cells. In one embodiment, the T cells are CD19-based CAR-T cells, which may be prepared by combination with any step of any method of preparing T cells for immunotherapy, such any method of preparation is incorporated herein by reference in whole by international publications 2015 / 120096 and 2017 / 070395 of the PCT Publications, both of which are incorporated herein by reference for the purpose of illustrating these methods; any method used for the preparation of Tisagenlecleucel / Kymriah®; any method used for the preparation of “ready-made” T cells for immunotherapy; and any other method of preparing lymphocytes for administration to humans. In some embodiments, the preparation process is adapted to specifically remove circulating tumor cells from cells obtained from a patient.

[0180] In one embodiment, the T cells are CD19 CAR-T cells prepared by the method described in international application PCT / US2016 / 057983. In one embodiment, a population of T cells depleted of circulating tumor cells is prepared from leukocyte apheresis products. These cells may be prepared as described in international application PCT / US2016 / 057983 and are further described herein as CD19 CAR-T cells. In short, CD19 CAR-T is an autologous CAR T cell product in which the T cells of interest are engineered to express a receptor consisting of a single-chain antibody fragment against CD19 linked to CD28 and CD3ζ activating domains, resulting in the removal of CD19-expressing cells. +Following the association of CARs with target cells, the CD3ζ domain activates a downstream signaling cascade that leads to the acquisition of effector functions such as T cell activation, proliferation, and cytotoxicity. The intracellular signaling domain of CD28 provides a co-stimulatory signal that functions with primary CD3ζ signaling to enhance T cell function, including interleukin (IL)-2 production. Together, these signals can stimulate the proliferation of CAR T cells and induce the death of target cells. Furthermore, activated T cells may secrete cytokines, chemokines, and other molecules that can mobilize and activate additional anti-tumor immune cells. Anti-CD19 CARs in CD19 CAR-T cells may include FMC63-28Z.

[0181] Because circulating tumor cells are present in certain cancers, the production of CD19 CAR-T cells is necessary. + and CD8 + The process includes a T cell enrichment step. The T cell enrichment or isolation step can reduce circulating CD19-expressing tumor cells in the leukocyte apheresis material and may be associated with the activation, proliferation, and depletion of anti-CD19 CAR T cells during production.

[0182] Methods described herein can improve the therapeutic outcomes or efficacy of immunotherapy or cell therapy, which may be adoptive T cell therapies selected from the group consisting of tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT®), allogeneic T cell transplantation, non-T cell transplantation, and any combination thereof. Adoptive T cell therapy broadly includes any method of selection, in vitro enrichment, and administration of the patient's autologous T cells or allogeneic T cells capable of recognizing and binding to tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, in vitro enriched, and administered to the patient. TIL cells may be either autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that includes isolating T cells capable of targeting tumor cells from a patient, enriching the T cells in vitro, and reintroducing the T cells to the same patient for administration. Allogeneic T cell transplantation may include the transplantation of ex vivo-grown spontaneously generated T cells or genetically engineered T cells. As described in more detail above, engineered autologous cell therapy is adoptive T-cell therapy, in which the patient's own lymphocytes are isolated, genetically modified to express tumor-targeting molecules, grown in vitro, and then returned to the patient for administration. Non-T cell transplantation may include autologous or allogeneic therapies using non-T cells, such as but not limited to natural killer (NK) cells.

[0183] The immunotherapy described in this application is engineered autologous cell therapy (eACT®). In this embodiment, the method may include collecting immune cells from a donor. The isolated immune cells may then be contacted with an exogenous activating agent (e.g., cytokine), grown, and engineered to express a chimeric antigen receptor ("engineered CAR T cells") or a T cell receptor ("engineered TCR T cells"). In some embodiments, the engineered immune cells treat a tumor in a subject. For example, one or more immune cells are transduced using a retrovirus containing heterologous genes encoding a cell surface receptor. In one embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell, for example, on the surface of a tumor cell. In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor. In another embodiment, one or more immune cells may be engineered to express a chimeric antigen receptor. The chimeric antigen receptor may include a binding molecule to a tumor antigen. The binding molecule may be an antibody or its antigen-binding molecule. For example, the antigen-binding molecule may be selected from scFv, Fab, Fab', Fv, F(ab')2, and dAb, as well as any fragments or combinations thereof. The chimeric antigen receptor may further include a hinge region. The hinge region may originate from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α. In one embodiment, the hinge region originates from the hinge region of IgG4. The chimeric antigen receptor may also include a transmembrane domain. The transmembrane domain may be the transmembrane domain of any transmembrane molecule that is a coreceptor on an immune cell or a transmembrane domain of a member of the immunoglobulin superfamily. In a particular embodiment, the transmembrane domain originates from the transmembrane domain of CD28, CD28T, CD8α, CD4, or CD19. In another embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain. In another embodiment, the transmembrane domain includes a domain derived from the CD28T transmembrane domain. The chimeric antigen receptor may further include one or more costimulatory signaling regions.For example, the co-stimulatory signaling region may be a signaling region for CD28, CD28T, OX-40, 41BB, CD27, inducible T cell costimulatory molecule (ICOS), CD3γ, CD3δ, CD3ε, CD247, Igα (CD79a), or Fcγ receptor. In a further embodiment, the co-stimulatory signaling region is a CD28 signaling region. In another embodiment, the co-stimulatory signaling region is a CD28T signaling region. In an additional embodiment, the chimeric antigen receptor further includes a CD3ζ signaling domain.

[0184] In some aspects, tumor antigens include 707-AP (707 alanine proline), AFP (α(a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen, β-catenin / m, β-catenin / mutant), BCMA (B cell maturation antigen), Bcr-abl (easily cleaved region-Abelson), CAIX (carbonic anhydrase IX), CD19 (surface antigen classification 19), CD20 (surface antigen classification 20), CD22 (surface antigen classification 22), CD30 (surface antigen classification 30), CD33 (surface antigen classification 33), and CD44v7 / 8 (surface antigen). Original classification 44, exon 7 / 8), CAMEL (CTL-recognizing antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27, variant), CDK4 / m (cyclin-dependent kinase 4, variant), CEA (carcinoembryonic antigen), CT (cancer / testis (antigen)), Cyp-B (cyclophyllin B), DAM (differentiation antigen, melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein Protein 40), Erbb2, 3, 4 (Erythroblastic leukemia virus oncogene homologs -2, -3, 4), ELF2M (Elongation factor 2, variant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate-binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (dicialoganglioside 2), GD3 (dicialoganglioside 3), GnT-V (N-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (H Recourse antigen), HER-2 / neu (human epithelial receptor-2 / neuronal, also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2, variant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit α-2), KIAA0205, KDR (kinase insertion domain receptor), κ-light chain, LAGE (L antigen),LDLR / FUT (Low-density lipid receptor / GDP-L-fucose: bD-galactosidase 2-αL-fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesoserine, mouse CMV-infected cells, MART-1 / Melan-A (melanoma antigen-1 recognized by T cells / melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin / m (myosin, variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous, variants 1, 2, 3), NA88-A (NA of patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York mammary gland differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferential expression antigen for melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal eccentric 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor-rejecting squamous antigen 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA The tumor antigen is selected from (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1), TPI / m (triose phosphate isomerase, variant), TRP-1 (tyrosinase-related protein 1, or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms oncogene), and any combination thereof. In one embodiment, the tumor antigen is CD19.

[0185] T-cell therapy involves administering engineered T cells expressing T cell receptors ("engineered TCR T cells") to a patient. The T cell receptor (TCR) may contain molecules that bind to tumor antigens. In some embodiments, tumor antigens include 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2 / neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIAA0205, KDR, κ-light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, mesoserine, mouse CMV-infected cells, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NKG2D ligand, NY-BR-1, NY-ESO-1, carcinoembryonic antigen, P15, p190 The following are selected from the group consisting of minor bcr-abl, Pml / RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF-R2, WT1, and any combination thereof.

[0186] "CD19-targeted gene-modified autologous T-cell immunotherapy" refers to a suspension of chimeric antigen receptor (CAR)-positive immune cells. An example of such immunotherapy is Clear CAR-T therapy, which uses CAR-T cells enriched with CD4+ / CD8+ T cells and does not contain circulating tumor cells. Another example is axi-cel® (also known as YESCARTA®). See Kochenderfer, et al., (J Immunother 2009;32:689 702). Other non-limiting examples include JCAR017, JCAR015, JCAR014, Kymriah (tisagenlecleucel), Uppsala U. anti-CD19 CAR (NCT02132624), and UCART19 (Celectis). See Sadelain et al. Nature Rev. Cancer Vol.3 (2003), Ruella et al., Curr Hematol Malig Rep., Springer, NY (2016), and Sadelain et al. Cancer Discovery (Apr 2013). For the preparation of CD19-targeted genetically modified autologous T cell immunotherapy, the patient's own T cells may be collected and genetically modified ex vivo by retroviral transduction to express a chimeric antigen receptor (CAR) containing a mouse anti-CD19 single-chain variable fragment (scFv) linked to the CD28 and CD3-ζ costimulatory domains. In some embodiments, the CAR contains a mouse anti-CD19 single-chain variable fragment (scFv) linked to the 4-1BB and CD3-ζ costimulatory domains. Anti-CD19 CAR T cells can be proliferated and returned to the patient by injection, where they can recognize and eliminate CD19-expressing target cells.

[0187] In one embodiment, the TCR includes a molecule that binds to a viral oncogene. In one embodiment, the viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (EBV), and human T lymphotropic virus (HTLV). In another embodiment, the TCR includes a molecule that binds to a testicular, placental, or fetal tumor antigen. In one embodiment, the testicular, placental, or fetal tumor antigen is selected from the group consisting of NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), and any combination thereof. In another embodiment, the TCR includes a molecule that binds to a lineage-specific antigen. In an additional embodiment, the group consisting of melanoma antigen-1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof, is selected from the group recognized by T cells. In certain embodiments, T cell therapy involves administering engineered CAR T cells to a patient that express a chimeric antigen receptor that binds to CD19 and further include a CD28 costimulatory domain and a CD3-ζ signaling region. In additional embodiments, T cell therapy involves administering KTE-C19 to a patient.In one embodiment, the antigenic portion further includes Epstein-Barr virus (EBV) antigen (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigen (e.g., VP1, VP2, VP3), hepatitis B virus antigen (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigen (e.g., encased glycoproteins E1 and E2), herpes simplex virus type 1, 2, or 8 (HSV1, HSV2, or HSV8) virus antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), and cytomegalovirus (C Examples include, but are not limited to, MV (Mobile Viral) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM or other envelope proteins), human immunodeficiency virus (HIV) virus antigens (glycoproteins gp120, gp41, or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) virus antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) virus antigens, or varicella-zoster virus antigens. In such embodiments, the cell surface receptor may be any TCR or any CAR that recognizes any of the aforementioned viral antigens on a target virus-infected cell. In other embodiments, the antigenic moiety is associated with cells having immune or inflammatory dysfunction.Examples of such antigenic moieties include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen involved in or associated with pathogenic autoimmune processes.

[0188] The methods disclosed herein may include T cell therapy, which involves transplanting one or more T cells into a patient. The T cells may be administered in a therapeutically effective dose. For example, the therapeutically effective dose of T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, may be at least about 10 4 Cells, at least about 10 5 Cells, at least about 10 6 Cells, at least about 10 7 Cells, at least about 10 8 Cells, at least about 10 9 , or at least about 10 10 This is possible. In another embodiment, the therapeutically effective dose of T cells, for example, engineered CAR+ T cells or engineered TCR+ T cells, is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 Cells, or about 10 8 These are cells. In one embodiment, the therapeutically effective dose of T cells, for example, engineered CAR+ T cells or engineered TCR+ T cells, is about 2 × 10⁻⁶ 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×106 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 Cells / kg, or approximately 9 x 10⁻⁶ 7 The value is cells / kg. In one embodiment, the amount of CD19 CAR-T cells is 2 × 10⁻⁶. 6 The unit is cells / kg, and the maximum dose for the target is 2 × 10 for a weight of 100 kg or more. 8 These are cells. In another embodiment, the amount of CD19 CAR-T cells is 0.5 × 10⁻⁶. 6 The dose is given per kg, and the maximum dose for the target group is 0.5 × 10⁶ for weights of 100 kg or more. 8 It is a cell.

[0189] Patients may be preconditioned or lymphocyte-depleted before administration of T-cell therapy. Patients may be preconditioned according to any method known in the art, including but not limited to treatment with one or more chemotherapeutic agents and / or radiotherapy. In some embodiments, preconditioning may include any treatment that is a reduction in the number of endogenous lymphocytes, elimination of cytokine sink, an increase in serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhancement of effector function of T cells administered after the treatment, enhancement of activation and / or utilization of antigen-presenting cells, or any combination thereof prior to T-cell therapy. Preconditioning may include increasing serum levels of one or more cytokines in the subject. The method further includes administering a chemotherapeutic agent. The chemotherapeutic agent may be a lymphocyte-depleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens, along with corresponding beneficial biomarkers, are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety. These are, for example, methods for conditioning patients requiring T-cell therapy, using a specified beneficial dose of cyclophosphamide (200 mg / m²). 2 / day~2000mg / m 2 ( / day) and the specified dose of fludarabine (20 mg / m²) 2 / day~900mg / m 2 The description includes administering ( / day) to the patient. Such a dosing regimen involves administering approximately 500 mg / m² to the patient before administering a therapeutically effective dose of genetically modified T cells to the patient. 2 Cyclophosphamide per day, and approximately 60 mg / m² 2 Treatment of the patient includes administering fludarabine daily for three days. In one embodiment, the pretreatment regimen includes cyclophosphamide 500 mg / m² over three days. 2 + Fludarabine 30 mg / m² 2These include: They may be administered on day -4, day -3, and day -2, or on day -5, day -4, and day -3 (day 0 is the day of cell administration). In one embodiment, the pretreatment regimen is cyclophosphamide 200 mg / m² 2 , 250 mg / m² 2 , 300 mg / m² 2 , 400V, 500mg / m³ 2 This is administered daily for 2, 3, or 4 days, with fludarabine 20 mg / m². 2 , 25 mg / m² 2 , or 30 mg / m² 2 This includes 2, 3, or 4 days. In one embodiment, pre-treatment chemotherapy (fludarabine 30 mg / m²) is administered after leukocyte apheresis. 2 / day and cyclophosphamide 500mg / m² 2The drug ( / day) is administered on days -5, -4, and -3, followed by intravenous infusion of a CD19 CAR-T cell suspension. In some embodiments, the intravenous infusion time is 15 to 120 minutes. In one embodiment, the intravenous infusion time is 1 to 240 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes. In some embodiments, the intravenous infusion time is up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 minutes. In some embodiments, the infusion volume is 50 to 100 mL. In some embodiments, the infusion volume is 20 to 100 mL. In some embodiments, the infusion volume is approximately 30, 35, 40, 45, 50, 55, 60, or approximately 65 mL. In some embodiments, the infusion volume is approximately 68 mL. In some embodiments, the suspension is frozen and used within 6, 5, 4, 3, 2, or 1 hour of thawing. In some embodiments, the suspension is not frozen. In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is agitated during infusion. In some embodiments, the immunotherapy is administered within 3 hours after thawing. In some embodiments, the suspension further comprises albumin. In some embodiments, albumin is present in an amount of about 2–3 volume%. In some embodiments, albumin is present in an amount of about 2.5 volume%. In some embodiments, albumin is present in an amount of about 1%, 2%, 3%, 4%, or 5% (v / v). In some embodiments, the albumin is human albumin. In some embodiments, the suspension further comprises DMSO. In some embodiments, DMSO is present in an amount of about 4–6 volume%. In some embodiments, DMSO is present in an amount of about 5 volume%. In some embodiments, DMSO is present in amounts of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v / v).

[0190] The methods disclosed herein may be used to treat cancer in a subject, to reduce tumor size, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to remove a tumor from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to induce remission in a patient, or any combination thereof. In certain embodiments, the methods can induce a complete response. In other embodiments, the methods can induce a partial response.

[0191] Cancers that can be treated include non-angiogenic tumors, tumors that are not yet substantially angiogenic, or tumors that are angiogenic. Cancers may also include solid or non-solid tumors.

[0192] In one embodiment, the method can be used to treat B-cell malignancies with high levels of circulating CD19-expressing tumor cells, which would be an appropriate treatment for a patient population characterized by a high level of unmet needs.

[0193] In some embodiments, CAR T cell intervention involves depleting circulating lymphoma cells and enriching CD4+ / CD8+ T cells by positive selection of mononuclear cells from leukocyte apheresis samples activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then proliferating from a T cell population transduced with a replication-deficient viral vector containing an anti-CD19 CAR construct. In some embodiments, the CAR construct is FMC63-28Z CAR. CAR T cells produced using this method may be referred to as KTE-X19. In some embodiments, the cells are self. In some embodiments, the cells are heterogeneous. In some embodiments, the dose of CAR-positive T cells is 2 × 10⁻⁶ 6 The dose is anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1 × 10⁶ 6 The dose is anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1.6 × 10⁶ 6 Anti-CD19 CAR T cells / kg, 1.8×10 6Anti-CD19 CAR T cells / kg, or 1.9 × 10⁻⁶ 6 The concentration is anti-CD19 CAR T cells / kg. In some embodiments, the CD19 CAR construct includes a CD3ζ T cell activation domain and a CD28 signaling domain.

[0194] In some embodiments, CAR T cells were administered at 25 mg / m² on days -5, -4, and -3 after leukocyte apheresis. 2 Fludarabine daily, and 900 mg / m² on day 2. 2 It is administered as a single infusion on day 0, following a daily cyclophosphamide pretreatment. In some embodiments, the pretreatment is 300 mg / m². 2 Cyclophosphamide daily, and 30 mg / m² for 3 days. 2 Contains fludarabine / day. In some embodiments, pre-treatment chemotherapy is 30 mg / m². 2 Fludarabine daily, plus 500 mg / m² on days -5, -4, and -3. 2 The formula contains cyclophosphamide per day. In some embodiments, the patient may also be administered acetaminophen and diphenhydramine or another H1 antihistamine about 30–60 minutes before anti-CD19 CAR T cell infusion. In some embodiments, the patient receives one or more additional doses of anti-CD19 CAR T cells.

[0195] In some embodiments, the MCL cancer is relapsed / refractory MCL (r / r MCL). In some embodiments, the patient has received one or more prior therapies. In some embodiments, the patient has received one to five prior therapies. In some embodiments, the prior therapies may include autologous SCT, anti-CD20 antibody, anthracycline or bendamustine-containing chemotherapy, and / or Bruton's tyrosine kinase inhibitors (BTKi). In some embodiments, the BTKi is ibrutinib (Ibr). In some embodiments, the BTKi is acalabrutinib (Acala). In some embodiments, this disclosure provides that MCL patients who have previously received ibrutinib have shown a more significant response to anti-CD19 CAR T-cell therapy compared to patients who have previously received acalabrutinib. Accordingly, this disclosure provides a method for treating r / r MCL with anti-CD19 CAR T cell therapy, wherein the patient has previously received ibrutinib or acalabrutinib treatment, and the cancer is preferably relapsed / refractory to that treatment. In some embodiments, the BTKi is tirabrutinib (ONO-4059), zanubrutinib (BGB-3111), CGI-1746, or spebratinib (AVL-292, CC-292).

[0196] In some embodiments, the present disclosure provides that for patients who received prior treatment with Ibr, Acala, or both, the median (range) of peak CAR T cell levels was 95.9 (0.4 - 2589.5), 13.7 (0.2 - 182.4), or 115.9 (17.2 - 1753.6), respectively. In some embodiments, the ORR / CR rates for anti-CD19 CAR T cell therapy in MCL patients were 94% / 65% in patients who received prior treatment with Ibr, 80% / 40% in patients who received prior treatment with Acala, and 100% / 100% in patients who received both BTKi treatments. In some embodiments, the 12-month survival rates in patients who received prior treatment with Ibr, Acala, or both were 81%, 80%, or 100%, respectively. In some embodiments, CAR T cell proliferation is related to the ORR / CR rate in patients who were previously treated with Ibr and / or Acala. Thus, in one embodiment, the patient is treated with both Ibr and Acala. In one embodiment, the present disclosure provides a method for predicting ORR / CR in MCL patients who were previously treated with Ibr and / or Acala by measuring peak CAR T cell levels and comparing them to a reference standard. In one embodiment, the present disclosure provides a method for predicting durable response based on the measurement of CAR T cell peak level / baseline tumor burden (CEN and INV). In one embodiment, the higher the ratio, the higher the likelihood of durable response at the 12-month time point / until. In one embodiment, a ratio of 0.00001 - 0.005 predicts non-response at the 12-month time point / until. In one embodiment, a ratio of 0.006 - 0.3 predicts recurrence at the 12-month time point / until. In one embodiment, a ratio of 0.4 - 1 predicts continued durable response at the 12-month time point / until. In one embodiment, the ratio can be determined by those skilled in the art from an average population.

[0197] In some embodiments, patients may receive bridging therapy (after leukocyte apheresis and before chemotherapy) with dexamethasone (e.g., 20-40 mg or equivalent PO or IV daily for 1-4 days), methylprednisolone, ibrutinib (e.g., 560 mg PO daily), and / or acalabrutinib (e.g., 100 mg PO twice daily), for example, completed within 5 days prior to pre-treatment chemotherapy. In some embodiments, such patients may have a high disease burden. In some embodiments, the bridging therapy is selected from immunomodulators, R-CHOP, bendamustine, alkylating agents, and / or platinum-based drugs.

[0198] In some embodiments, this disclosure provides that all MCL patients who responded to CAR T cell infusion achieved T cell proliferation, while proliferation was not observed in non-responding patients. In some embodiments, the response is the objective response rate (complete response + partial response). This disclosure shows that CAR T cell levels correlate with the ORR for the first 28 days and the area under the curve (AUC) from day 0 to 28. 0-28 ) and peak levels were more than 200 times higher in responders compared to non-responders, which is due to minimal residual disease (MRD, sensitivity 10 -5 As indicated by peak / AUC CAR T cell levels that are more than 80 times higher in MRD-positive patients (at 4 weeks) compared to MRD-positive patients (at 4 weeks) in the negative case, greater proliferation suggests a better and possibly more meaningful response. Therefore, this disclosure provides a method for predicting patient response and MRD to CAR T cell therapy for MCL, comprising measuring peak / AUC CAR T cell levels and comparing them to a reference standard. In some embodiments, peak CAR T cell proliferation is observed 8 to 15 days after CAR T cell administration. In some embodiments, CAR T cell levels are measured by qPCR. In some embodiments, peak CAR T cell levels, AUC 0-28、and / or MRD is monitored by next-generation sequencing. In some examples, the number of CAR T cells is measured as the number of cells in 1 microliter of blood. In some examples, the number of CAR T cells is measured by the copy number of the CAR gene in 1 μg of host DNA. In some examples, the number of CAR T cells is measured as described in Kochenderfer J.N et al. J.Clin.Oncol. 2015;33:540-549. In one embodiment, the CAR T cell level is measured as described in Locke FL et al. Mol Ther. 2017;25(1):285-295.

[0199] In some embodiments, the present disclosure provides that CAR T cell proliferation was higher in grade ≥3 MCL patients than in those with grade ≤3 CRS and NE events. Thus, the present disclosure provides a method for predicting grade ≥3 CRS and NE events, including measuring CAR T cell proliferation after CAR T cell therapy and comparing the level to a reference value, wherein the higher the CAR T cell proliferation, the higher the likelihood of grade ≥3 CRS and NE events.

[0200] In some embodiments, cytokine levels are measured by protein or mRNA levels (either). In some embodiments, cytokine levels are measured as described in Locke FL et al. Mol Ther. 2017;25(1):285-295.

[0201] In some embodiments, the present disclosure provides that the serum GM-CSF and IL-6 peak levels (reached approximately 8 days after CAR T cell administration) were positively associated with grade ≥3 CRS and grade ≥3 NE in MCL patients. Thus, the present disclosure provides a method for predicting grade ≥3 CRS and grade ≥3 NE, including measuring the peak levels of GM-CSF and IL-6 after CAR T cell administration and comparing them to reference levels, wherein the higher the peak levels of these cytokines, the higher the likelihood of grade ≥3 CRS and NE.

[0202] In some embodiments, the Disclosure provides that in MCL patients, serum ferritin levels were positively associated with grade ≥ 3 CRS. Accordingly, the Disclosure provides a method for predicting grade ≥ 3 CRS, comprising measuring peak serum ferritin levels after CAR T cell administration and comparing them to a reference level, where higher peak ferritin levels indicate a higher likelihood of grade ≥ 3 CRS.

[0203] In some embodiments, the Disclosure provides that in MCL patients, serum IL-2 and IFNγ were positively associated with grade ≥ 3 NE. Accordingly, the Disclosure provides a method for predicting grade ≥ 3 CRS, comprising measuring the peak levels of serum IL-2 and IFNγ after CAR T cell administration and comparing them to reference levels, wherein higher peak levels of IL-2 and IFNγ indicate a higher probability of grade ≥ 3 NE.

[0204] In some embodiments, the Disclosure provides that in MCL patients, cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and vascular cell adhesion molecules (VCAMs) were positively associated with grade ≥ 3 NEs. Accordingly, the Disclosure provides a method for predicting grade ≥ 3 CRS, comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecules (VCAMs) after CAR T cell administration and comparing them to reference levels, wherein higher cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecules (VCAMs) are associated with a higher likelihood of grade ≥ 3 NEs.

[0205] In some embodiments, the disclosure provides that peak serum levels of cytokines positively associated with grade ≥ 3 CRS include IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and perforin. In some embodiments, the disclosure provides that peak serum levels of cytokines associated with grade 3 or higher NE include IL-2, IL-1Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. In some embodiments, the disclosure provides that cytokines associated with both grade 3 or higher CRS and NE include IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B. In some embodiments, cytokine serum levels peak within 7 days of CAR T cell administration. Accordingly, the Disclosure provides a method for predicting grade ≥ 3 CRS after CAR T cell administration, comprising measuring peak serum levels of IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and / or perforin after anti-CD19 CAR T therapy and comparing those levels to a reference standard. Accordingly, the Disclosure also provides a method for predicting grade ≥ 3 CRS and grade ≥ 3 NE in MCL, comprising measuring peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B after anti-CD19 CAR T therapy and comparing those levels to a reference standard.

[0206] In some embodiments, the disclosure provides that in MCL patients with mutant TP53 versus wild-type TP53, there was a tendency for increased peak cytokine levels of proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1). Accordingly, in some embodiments, the disclosure provides methods for improving the response to CAR T cell therapy in MCL, including manipulating levels of proliferative and / or inflammatory cytokines after CAR T cell administration.

[0207] In some embodiments, the disclosure provides that patients who were MRD-negative one month after CAR T cell administration showed a tendency toward increased peak levels of IFN-γ and IL-6, as well as increased levels of IL-2, compared to patients who were MRD-positive one month after administration. Accordingly, the disclosure provides a method for predicting whether a patient will be MRD-negative in MCL, comprising measuring peak serum levels of IFN-γ, IL-6, and / or IL-2 after anti-CD19 CAR T therapy and comparing those levels to a reference standard.

[0208] In some embodiments, this disclosure aims to provide a T cell product in which circulating lymphoma cells are depleted, CD4+ / CD8+ T cells are enriched by positive selection of mononuclear cells from a leukocyte apheresis sample activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then T cells are proliferated from a T cell population transduced with a replication-deficient viral vector containing an anti-CD19 CAR construct. In some embodiments, such a T cell product may be used to treat ALL, CLL, and AML. In some embodiments, the CAR construct is FMC63-28Z CAR. In some embodiments, the cells are autologous. In some embodiments, the cells are heterologous. In some embodiments, the dose of CAR-positive T cells is 2 × 10⁻⁶ 6 The dose is anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1 × 10⁶ 6 The dose is anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1.6 × 10⁶6 Anti-CD19 CAR T cells / kg, 1.8×10 6 Anti-CD19 CAR T cells / kg, or 1.9 × 10⁻⁶ 6 The product is anti-CD19 CAR T cells / kg. In some embodiments, the CD19 CAR construct includes a CD3ζ T cell activation domain and a CD28 signaling domain. In some embodiments, the T cell product is KTE-X19. In some embodiments, this disclosure provides that an anti-CAR T cell product prepared as described in the preceding paragraph may be used in B cell ALL and B cell NHL. In some embodiments, product characteristics may be selected from a percentage of T cells of a specific subset (naive, central memory, effector, and effector memory), a percentage of CD4+ cells, a percentage of CD8+ cells, and a CD4 / CD8 ratio. In some embodiments, the product characteristic is the level of IFNγ production (pg / mL) in co-culture with target CD19-expressing cancer cells (e.g., Toledo) mixed in a 1:1 ratio with anti-CD19 CAR T product cells. In one embodiment, IFNγ may be measured in cell culture medium 24 hours after incubation using a qualified ELISA. In some embodiments, one or more of these product characteristics are superior to those of anti-CAR T cells prepared from leukocyte apheresis without CD4+ / CD8+ positive cell enrichment. In some embodiments, the superior product characteristics may be selected from an increased percentage of cells with a naive phenotype (CD45RA+CCR7+), a decreased percentage of cells with a differentiated phenotype (CCR7-), a decreased level of IFNγ-producing cells, or an increased level of CD8+ cells. In some embodiments, the anti-CD19 T cell product is T CM Central memory T cells (CD45RA-CCR7+); T EFF Effector T cells (CD45RA+CCR7-); T EM , effector memory T cells (CD45RA-CCR7-); and / or T N, containing naive-like T cells (CD45RA+CCR7+). In some embodiments, the product is CD45RA+CCR7+, meaning T cells containing stem-like memory cells. N It contains naive-like T cells. In some embodiments, the T cell product is KTE-X19. In some embodiments, KTE-X19 has IFNγ production of 190 pg / mL or more. In certain embodiments, KTE-X19 has ≥90% CD3+ cells. In some other embodiments, the percentage of NK cells in KTE-X19 is 0.1% (ranging from 0.0% to 2.8%). In some additional embodiments, CD3 in KTE-X19 - The percentage of cellular impurities is 0.5% (ranging from 0.3% to 3.9%).

[0209] In some embodiments, the cancer is relapsed / refractory B-cell ALL. In some embodiments, the patient is 21 years of age or younger. In some embodiments, the patient is 21 years of age or younger, weighs ≥10 kg, and has B-cell ALL that, at least 100 days prior to registration, is resistant to primary treatment, relapsed within 18 months of initial diagnosis, R / R after two or more systemic therapies, or R / R after allogeneic stem cell transplantation. In one embodiment, the cancer is low-grade lymphoma or leukemia. In one embodiment, the cancer is intermediate-grade B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), mantle cell lymphoma and its blastoid variants, and many types, subtypes, and variants of B-lymphoblastic lymphoma. DLBCL can be DLBCL NOS, T-cell / histiocyte-rich large B-cell lymphoma, primary CNS DLBCL, primary cutaneous DLBCL (lower extremity type), or EBV-positive DLBCL in the elderly. Other large B-cell lymphomas include primary mediastinal (thymic) LBCL, DLBCL associated with chronic inflammation, lymphomatous granulomatosis, ALK-positive LBCL, plasmablastic lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, and primary coelomic lymphoma. Other types of lymphoma include unclassified B-cell lymphoma with features intermediate between DLBCL and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin lymphoma, splenic marginal zone B-cell lymphoma, MALT type extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma B-cell lymphoma, hairy cell leukemia, lymphoplasmacytic lymphoma (Waldenström hypergammaglobulinemia), Richter transformation, and primary coelomic fluid lymphoma. The cancer may be at any stage from stage 1 to stage 4.

[0210] In some embodiments, the pre-treatment chemotherapy / lymphocyte depletion regimen is administered after a washout of bridging chemotherapy for ≥7 days or 5 × half-life (if shorter). In some embodiments, the pre-treatment chemotherapy / lymphocyte depletion regimen is administered as fludarabine intravenously (IV) 25 mg / m² on days -4, -3, and -2. 2Cyclophosphamide IV 900 mg / m² on day 1 and day 2. 2 The regimen consists of one day. On day 0, a single infusion of anti-CD19 CAR T cells may be administered. In some embodiments, an additional infusion of anti-CD19 CAR T cells may be administered thereafter. In some embodiments, if a patient achieves a complete response to the initial infusion and experiences a progression after more than 3 months of remission, a second infusion of anti-CD19 CAR T cells may be administered, provided that CD19 expression is maintained and there is no suspicion of neutralizing antibodies against CAR.

[0211] In some embodiments, droplet digital polymerase chain reaction can be used to measure the presence, proliferation, and persistence of transduced anti-CD19 CAR T cells in the blood. In some embodiments, the procedure is described in Locke FLet al. Mol Ther. 2017;25(1):285-295. In some embodiments, this disclosure provides that CAR T cells may be undetectable at relapse. The median peak CAR T cell level is 1 × 10⁻⁶. 6The highest values ​​may be at individual CAR T cells / kg, and may be similar between patients receiving original AE management and those receiving revised AE management. In some embodiments, patients achieving CR / CRi had higher median peak proliferation than non-responders, and similarly, patients with undetectable vs. detectable MRD. Higher peak proliferation values ​​were also observed in patients with grade ≥3 NE vs. grade ≤2NE. Some relapsing patients may or may not have detectable CD19-positive cells at the time of relapse. In some embodiments, undetectable MRD, defined as less than one leukemia cell per 10,000 viable cells, can be assessed using flow cytometry (NeoGenomics, Fort Myers, Florida) according to the methods described in Borowitz MJ, Wood BL, Devidas M, et al. Blood. 2015;126(8):964-971; Bruggemann M. et al. Blood Adv. 2017;1(25):2456-2466; or Gupta S. et al. Leukemia. 2018;32(6):1370-1379.

[0212] In some embodiments, the disclosure provides that peak serum IL-15 levels are lower in patients with grade ≥ 3 CRS. In some embodiments, the disclosure provides that the median peak levels of several pro-inflammatory markers tend to be higher in patients with grade 3 or higher CRS and patients with grade 3 or higher NE (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, granzyme B). Accordingly, in some embodiments, the disclosure provides a method for predicting whether a patient will have grade 3 or higher CRS by measuring the peak level of serum IL-15 and comparing it to a reference standard. In some embodiments, the disclosure provides a method for predicting whether a patient will have grade ≥ 3 CRS and / or grade ≥ 3 NE by measuring the peak levels of IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, and / or granzyme B and comparing them to a reference standard. In some embodiments, the present disclosure provides a method for improving anti-CD19 CAR T cell therapy by administering a drug that reduces the level of one or more of these biomarkers.

[0213] Reference levels / standards can be established by any method known to those skilled in the art. They function to identify groups of thresholds or values ​​(e.g., quartiles), from which comparisons can be made to determine which groups, or thresholds above or below, each measure (cytokine level, CAR T cell count, etc.) each subject includes. These groups are established from comparisons of different populations selected to be typical in the art. Depending on where the measure is included, several therapeutic properties, such as objective response, CRS grade, NE grade, etc., can be predicted.

[0214] In certain embodiments, cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma, atypical teratomatoid / rhabdomyosarcoma-like tumors, central nervous system cancers, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, central nervous system cancers, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), and chronic bone marrow cancer. Leukemia luteum (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germ cell tumors, central nervous system cancer, endometrial cancer, ependymoblastoma, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, eye cancer, malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcoma, germ cell tumors, gestational trophoblastoma, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, histiocytosis Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic islets), Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with occult primary midline cancer associated with the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplasia Myelodysplastic / myeloproliferative neoplasms, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal and nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropneumoblastoma,The cancers that can be selected from pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström type macroglobulinemia, and tumors derived from Wilms tumor. In certain embodiments, the cancer is treated with KTE-X19.

[0215] In one embodiment, the method may be used to treat a tumor, which is a lymphoma or leukemia. Lymphomas and leukemias are cancers of the blood that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell in the bone marrow. These stem cells produce both myeloid progenitor cells and lymphoid progenitor cells, which in turn give rise to the various types of white blood cells found in the body. White blood cells that arise from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells that arise from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient. In a particular embodiment, the tumor is treated with KTE-X19.

[0216] Generally, lymphomas can be divided into at least two subgroups: Hodgkin lymphomas and non-Hodgkin lymphomas. Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of cancers originating from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B-cell lymphomas account for 80–85% of reported cases. In 2013, it was estimated that there were approximately 69,740 new cases of NHL and over 19,000 disease-related deaths. Non-Hodgkin lymphomas are the most common hematological malignancies, the seventh leading site of new cancers in men and women, accounting for 4% of new cancer cases and 3% of cancer-related deaths. In certain embodiments, lymphomas are treated with KTE-X19.

[0217] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. In the United States, approximately 22,000 people are newly diagnosed with DLBCL each year. It is classified as an intermediate-grade lymphoma, with the majority of patients cured with conventional chemotherapy (NCCN Guidelines, NHL 2014). First-line therapy for DLBCL typically involves an anthracycline-containing regimen including rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of approximately 80% and a complete response rate of approximately 50%, with about one-third of patients being resistant to initial treatment or relapsing after R-CHOP. Of patients who relapse after responding to first-line therapy, approximately 40–60% can achieve a second response with additional chemotherapy. Standard second-line therapy for patients eligible for autologous stem cell transplantation (ASCT) includes rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), with objective response rates of approximately 63% and complete response rates of approximately 26%, respectively. Patients who respond to second-line therapy and are deemed well-suited for transplantation receive consolidation therapy with high-dose chemotherapy and ASCT, which is curative in about half of transplant patients. Patients who fail ASCT have a very poor prognosis and there are no curative options. Primary mediastinal large B-cell lymphoma (PMBCL) has different clinical, pathological, and molecular characteristics compared to DLBCL. PMBCL is thought to originate from thymic (medullary) B cells and accounts for about 3% of patients diagnosed with DLBCL. PMBCL typically occurs in a young adult population in their 30s and is slightly more common in women. Gene expression profiling suggests a disregulated pathway in PMBCL that overlaps with that of Hodgkin lymphoma.Initial treatment for PMBCL generally involves anthracycline-containing regimens, including rituximab, with or without regional irradiation, such as modified doses of etoposide, doxorubicin, and cyclophosphamide, along with vincristine, prednisone, and rituximab (DA-EPOCH-R). Follicular lymphoma (FL) and B-cell lymphoma are the most common low-grade (delayed growth) forms of NHL, accounting for approximately 20-30% of all NHL cases. Some patients with FL undergo histological transformation to DLBCL (TFL), which is more aggressive and associated with poor outcomes. Histological transformation to DLBCL occurs at a rate of approximately 3% per year over 15 years, after which the risk of transformation continues to decrease. The biological mechanism of histological transformation is unknown. Initial treatment for TFL is influenced by prior treatment for follicular lymphoma, but generally involves anthracycline-containing regimens with rituximab to eliminate the intermediate-grade portion of the disease. Treatment options for relapsed / refractory PMBCL and TFL are similar to those for DLBCL. Given the low prevalence of these diseases, large-scale prospective randomized trials have not been conducted in these patient populations. Patients with chemotherapy-resistant disease have a similar or worse prognosis than those with refractory DLBCL. For example, subjects with refractory intermediate-grade NHL (e.g., DLBCL, PMBCL, and TFL) have high unmet medical needs, and further research with new therapies is needed in these populations. In certain embodiments, DLBCL is treated with KTE-X19.

[0218] The CAR T cell therapy described herein may be administered as a first-line therapy or a second-line or later therapy. In some embodiments, the CAR T cell therapy may be administered as a third-line therapy, fourth-line therapy, fifth-line therapy, etc. The preferred prior treatment may be any prior anticancer therapy, but is not limited to, Bruton's tyrosine kinase inhibitors (BTKi), checkpoint inhibitors (e.g., anti-PD1 antibodies such as pembrolizumab (Keytruda), cemiprimab (ribtayo), and nivolumab (Opdivo); anti-PD-L1 antibodies such as atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi); anti-CTLA-4 antibody such as ipilimumab (Yervoy)), anti-CD19 antibodies (e.g., blinatumomab), anti-CD52 antibodies (e.g., allentuzumab); allogeneic stem cell transplantation, anti-CD20 antibodies (e.g., rituximab), systemic chemotherapy, rituximab, anthracyclines, ofatumumab, and combinations thereof. The prior treatment may also be performed in combination with the CD19 CAR T therapy of this application. In one embodiment, eligible patients may have a disease that is resistant to the most recent treatment or may have relapsed within one year after autologous hematopoietic stem cell transplantation (HSCT / ASCT). CAR T-cell therapy may be administered to patients who have or are suspected of having cancer that is resistant to one or more prior therapies and / or has subsequently relapsed. The cancer may be resistant to first-line therapy (i.e., primary treatment resistant) or resistant to one or more treatment options. The cancer may have relapsed 12 months after initial remission, relapsed or become resistant after two or more prior therapies, or relapsed after HSCT / ASCT. In some embodiments, the cancer is resistant to ibrutinib or acalabrutinib. In some embodiments, the cancer is NHL, and the disease must be primary treatment resistant, R / R after two or more systemic therapies, or R / R after autologous or allogeneic stem cell transplantation at least 100 days prior to enrollment in CAR T-cell therapy and at least 4 weeks after discontinuing immunosuppressant therapy. In certain embodiments, the CAR T cell therapy is KTE-X19.

[0219] Therefore, this method may be used to treat lymphoma or leukemia, which are B-cell malignancies. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytic B-cell lymphoma), splenic, diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma. In some embodiments, lymphoma or leukemia is B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasmacytoma (e.g., plasmacytoma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicular central lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, A LK+ large B-cell lymphoma, plasmablastic lymphoma, primary coelomic lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, Burkitt lymphoma / leukemia, T-cell pre-lymphocytic leukemia, T-cell macrogranular lymphocytic leukemia, rapidly progressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma The following are selected from peritoneum, blastic NK cell lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papular dysplasia, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T-lymphoblastic leukemia / lymphoma, and Hodgkin lymphoma. In some embodiments, the cancer is resistant to one or more prior therapies and / or has relapsed after one or more prior therapies.In certain embodiments, leukemia or lymphoma is treated with KTE-X19.

[0220] In one embodiment, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In other embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is resistant to one or more of chemotherapy, radiation therapy, immunotherapy (including T cell therapy and / or treatment with an antibody or antibody-drug conjugate), autologous stem cell transplantation, or any combination thereof, or the cancer has recurred subsequently. In one embodiment, the cancer is refractory diffuse large B-cell lymphoma. In certain embodiments, the cancer is treated with KTE-X19.

[0221] In some embodiments, the CAR T cell therapy is KTE-X19 and the cancer is selected from MCL, ALL, CLL, and SLL. In some embodiments, the CAR T cell therapy is KTE-X19 and the cancer is NHL. In some embodiments, the cancer is selected from diffuse large B-cell lymphoma NOS (DLBCL NOS), primary mediastinal large B-cell lymphoma, Burkitt lymphoma (BL), Burkitt-like lymphoma, or intermediate B-cell lymphoma unclassified between DLBCL and BL. In some embodiments, the cancer is recurrent / refractory. In some embodiments, the KTE-X19 treatment is administered as a first-line therapy, a second-line therapy, or after one or more prior therapies. In some embodiments, the patient is a pediatric patient, an adolescent patient, an adult patient, under 65 years old, over 65 years old, or any other age group.

[0222] In some embodiments, the immune cell-containing compositions disclosed herein may be administered in combination with any number of additional therapeutic agents. In one embodiment, the additional therapeutic agents are administered concurrently with T-cell therapy. In one embodiment, the additional therapeutic agents are administered before, during, and / or after T-cell therapy. In one embodiment, one or more additional therapeutic agents are administered prophylactically. In one aspect, the immune cell-containing compositions are administered together with agents for managing adverse events (many of which are described elsewhere in this application, including in the Examples section). These medications can manage signs and symptoms of one or more adverse reactions, such as fever, hypotension, tachycardia, hypoxia, and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0223] Examples of such drugs include, but are not limited to, tocilizumab, steroids (e.g., methylprednisolone), and rabbit antithymocyte globulin. In some embodiments, vancomycin and aztreonam (1 gm IV each, twice daily) may be administered for nonneutrophilic fever. In some embodiments, the method further includes administering a non-sedating antiseizure agent for seizure prevention, administering at least one of erythropoietin, darbepoetin alfa, platelet transfusion, filgrastim, or pegfilgrastim, and / or administering tocilizumab, siltuximab. In one embodiment, the drug is a member of the CSF family, such as GM-CSF (granulocyte-macrophage colony-stimulating factor, also known as CSF2). GM-CSF can be produced by several hematopoietic and non-hematopoietic cell types upon stimulation, activating / priming the bone marrow population to produce inflammatory mediators such as TNF and interleukin-1β (IL1β). In some embodiments, the GM-CSF inhibitor is an antibody that binds to and neutralizes circulating GM-CSF. In some embodiments, the antibody is selected from rengilumab, namilumab (AMG203); GSK3196165 / MOR103 / Otilimab (GSK / MorphoSys), KB002 and KB003 (KaloBios), MT203 (Micromet and Nycomed), and MORAb-022 / gimsilumab (Morphotek). In some embodiments, the antibody is its biosimilar. In some embodiments, the antagonist is E21R, a modified form of GM-CSF that antagonizes the function of GM-CSF. In some embodiments, the inhibitor / antagonist is a small molecule. In one embodiment, the CSF family member is M-CSF (also known as macrophage colony-stimulating factor or CSF1). Non-exclusive examples of agents that inhibit or antagonize CSF1 include small molecules, antibodies, chimeric antigen receptors, fusion proteins, and other agents. In one embodiment, the CSF1 inhibitor or antagonist is an anti-CSF1 antibody.In one embodiment, the anti-CSF1 antibody is selected from those produced by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110 / lanotuzumab), or any one of these biosimilar versions. In some embodiments, the inhibitor or antagonist inactivates the activity of either the GM-CSF-R-alpha (also known as CSF2R) or CSF1R receptor. In some embodiments, the inhibitors include maprilimumab (formerly CAM-3001), a fully human GM-CSF receptor α monoclonal antibody currently under development by MedImmune, Inc.; kabilizumab (Five Prime Therapeutics); emactuzumab, also known as LY3022855 (IMC-CS4) (Eli Lilly), RG7155, or RO5509554; FPA008, a humanized mAb (Five Prime / BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041 / AFS98 / TG3003 (ElsaLys Bio, Transgene); and SNDX-6352 (Syndax). In some embodiments, the inhibitor or antagonist is expressed in CAR-T cells.In some embodiments, the inhibitor is a small molecule (e.g., heteroarylamide, quinolinone, pyridopyrimide; BLZ945 Novartis), PLX7486, ARRY-382, pexidiltinib (also known as PLX3397), or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridine-3-yl)methyl)-N-06-(trifluoromethyl)pyridine-3-yl)methyl)pyridine-2-amine; GW 2580 (CAS 870483-87-7), Ki20227 (CAS 623142-96-1), AC708 by Ambit Siosciences, or Cannarile et al. Journal for ImmunoTherapy of Cancer Any CSF1R inhibitor listed in 2017, 5:53 and U.S. Patent Application Publication No. 20180371093 (the inhibitors disclosed herein are incorporated herein by reference). Additional neutralizing antibodies against GM-CSF or its receptor are described, for example, in the techniques contained in “GM-CSF as a target in inflammatory / autoimmune disease: current evidence and future therapeutic potential” Hamilton, JA Expert Rev. Clin. Immunol., 2015; and “Targeting GM-CSF in inflammatory diseases” Wicks, IP, Roberts, AWNat. Rev. Rheumatol., 2016. In other embodiments, the agent is an anti-IL6 or anti-IL-6 receptor blocker, including tocilizumab and siltuximab.

[0224] In one embodiment, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocon, metredopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, ethyleneimines and methylameramamines including trimethylomellamine regimen; nitrogen mustards, e.g., chlorambucil, chlorna Fadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kakutinomycin, calicheamicin, carabicin, carminomycin, cardiomycin Nofilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, pofilomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluoro Uracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Anti-adrenaline agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatrexate; defofamine; demecoltin; diazicone; elformitin, eriptinium acetate; etogluside; gallium nitrate; hydroxyurea, lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; Pirarubicin; podophyllic acid, 2-ethylhydrazide; procarbazine; PSK®, razoxane, schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine, 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C, mitoxantrone; vincristine, vinorelbine, navelbine, novantrone; teniposide; daunomycin; aminopterin; xeroda, ibandronate; CPT-11, topoisomerase inhibitor RFS2000;Examples include retinoic acid derivatives such as difluoromethylomitin (DMFO), Targretin (trademark) (bexarotene), Panretin (trademark) (allitretinoin), ONTAK (trademark) (denileukin difuticox), esperamycin, capecitabine, and any of the above pharmaceutically acceptable salts, acids, or derivatives. In some embodiments, compositions comprising CAR and / or TCR-expressing immunoeffector cells disclosed herein may be administered in combination with anti-estrogens acting to modulate or inhibit hormonal effects on tumors, such as anti-estrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salt, acid, or derivative thereof. Combinations of chemotherapeutic agents including CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, may also be administered as appropriate.

[0225] The (chemo)therapy agent may be administered concurrently with the administration of the engineered cells or nucleic acids, or within one week after administration. In other embodiments, the (chemo)therapy agent is administered for 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cells or nucleic acids. In some embodiments, the (chemo)therapy agent is administered at least one month before the administration of the cells or nucleic acids. In some embodiments, the method further includes the administration of two or more chemotherapeutic agents.

[0226] Various additional therapeutic agents may be used in combination with the compositions or drugs / treatments described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche), tocilizumab (with and without corticosteroids), inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R (anti-CSF1 antibodies manufactured by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110 / lacnotuzumab), mabrilimumab (formerly CAM-3001), and a fully human GM-CSF receptor α monoclonal antibody currently under development by MedImmune, Inc.; and kabilizumab (Five Prime). Examples include: Therapeutics; emactuzumab, also known as LY3022855 (IMC-CS4) (Eli Lilly), RG7155, or RO5509554; FPA008, humanized mAb (Five Prime / BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041 / AFS98 / TG3003 (ElsaLys Bio, Transgene); SNDX-6352 (Syndax). In some embodiments, the inhibitor or antagonist is expressed in CAR-T cells.In some embodiments, the inhibitor is a small molecule (e.g., heteroarylamide, quinolinone, pyridopyrimide; BLZ945 Novartis), PLX7486, ARRY-382, pexidiltinib (also known as PLX3397), or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridine-3-yl)methyl)-N-06-(trifluoromethyl)pyridine-3-yl)methyl)pyridine-2-amine; GW 2580 (CAS 870483-87-7), Ki20227 (CAS 623142-96-1), AC708 by Ambit Siosciences, or Cannarile et al. Journal for ImmunoTherapy of Cancer Any CSF1R inhibitor listed in 2017,5:53 and U.S. Patent Application Publication No. 20180371093 (the inhibitors disclosed herein are incorporated herein by reference). Additional neutralizing antibodies against GM-CSF or its receptor are described in the art.Additional therapeutic agents suitable for use in combination with the compositions or agents / treatments and methods disclosed herein include ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), and trastuzumab emtansine (KADCYL). A(registered trademark), Imatinib (GLEEVEC(registered trademark)), Cetuximab (ERBITUX(registered trademark)), Panitumumab (VECTIBIX(registered trademark)), Katsumakisomab, Ibritumomab, Ofatumumab, Tositumomab, Brentuximab, Alemtuzumab, Gemtuzumab, Erlotinib, Gefitinib, Vandetanib, Afatinib, Lapatinib, Neratinib, Lenalidomide, Axitinib, Macitinib Pazopanib, sunitinib, sorafenib, tocilizumab, toceranib, restaurtinib, axitinib, cejiranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, carbozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, restaurtinib, ruqi Examples of mTOR inhibitors include, but are not limited to, solitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukindiffitox, everolimus, and temsirolimus; hedgehog inhibitors such as sonedegib and bismodegib; and CDK inhibitors such as palbociclib.

[0227] Compositions or agents / therapeutic agents containing immune cells are administered or may be administered together with anti-inflammatory agents. Examples of anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, corticosteroids, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, and tramadol (proporxiphene hydrochloride). Examples of glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Examples of biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Examples of biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (Auranofin) and intramuscular), and minocycline.

[0228] The compositions or agents / therapeutic agents described herein may be administered in combination with cytokines and / or cytokine modulators as additional therapeutic agents. Examples of cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin; glycoprotein hormones, e.g., follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone. hormone (LH), hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin, placental lactogen, murelan inhibitor; mouse gonadotropin-related peptide; inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF), e.g., NGF-beta; platelet growth factor; transforming growth factor (TGF), e.g., TGF-α and TGF-β; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO, Epogen®, Procrit®); bone induction factor, inter These include interferons, e.g., interferon-alpha, beta, and gamma; colony-stimulating factors (CSFs), e.g., macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), e.g., IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15; tumor necrosis factor, e.g., TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, and bioactive equivalents of naturally occurring cytokines. In one embodiment, the compositions described herein are administered together with steroids or corticosteroids.

[0229] Corticosteroid therapy may be used to treat adverse events. Corticosteroids (or any other steroid, and any other treatment for adverse events) may be used prophylactically before symptoms of any adverse event are detected, and / or after the detection of any adverse event. They may be administered at least one day before T-cell administration, on the day of T-cell administration (before, after, and / or during T-cell administration), and / or after T-cell administration. They may be administered before, during, or after pretreatment. Any corticosteroid may be suitable for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, these two are administered in combination. In some embodiments, the glucocorticoids include synthetic and non-synthetic glucocorticoids. Examples of glucocorticoids include alclomethasone, alginate, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasol, crocoltron (e.g., crocoltron pivalate), cloprednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortibazole, deflazacol, desonide, desoxymethasone, and dexamethasone (e.g., dexamethasone phosphate). 21. Dexamethasone acetate, dexamethasone sodium phosphate), diflorazone (e.g., diflorazone diacetate), diflucortalone, difluprednate, enoxolone, fluazacol, flucuronide, fludrocortisone (e.g., fludrocortisone acetate), flumetazone (e.g., flumetazone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, flucortin, flutrolone, fluorometholone (e.g., fluorometholone acetate), fluperolone (e.g., fluperone acetate), flupredniden, fluprednisolone, flulandrenolide, fluticasone (e.g., fluticasone propionate), formocortal,Halcinonide, halobetasol, halomethasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone butate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemysuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate, mazipredone, medrisone, meprednisone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemysuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), paramethasone (e.g., paramethasone acetate) Tazone), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemysuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulfobenzoic acid, prednisolone stearate, pre Examples include, but are not limited to, donisolone tebutate (prednisolone tetrahydrophthalate), prednisone, prednival, prednylidene, rimexolone, thixocortol, and triamcinolone (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are described in detail, for example, Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980) and Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) and any other editions.These are incorporated herein by reference. In some embodiments, the glucocorticoid is selected from cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a mineralocorticoid. Any other steroid may be used in the manner provided herein.

[0230] One or more corticosteroids may be administered in any dose and frequency that is appropriate for the severity / grade of adverse events (e.g., CRS and NE). In another embodiment, the administration of corticosteroids includes oral or intravenous administration of 10 mg of dexamethasone 1 to 4 times per day. Another embodiment, sometimes referred to as “high-dose” corticosteroids, includes intravenous administration of methylprednisone 1 g / day alone or in combination with dexamethasone. In some embodiments, one or more corticosteroids are administered in doses of 1 to 2 mg / kg per day.

[0231] Corticosteroids may be administered in any amount effective in improving one or more symptoms associated with adverse events such as CRS or neurotoxicity. Corticosteroids, such as glucocorticoids, can be administered to an adult weighing 70 kg in doses of approximately 0.1-100 mg, 0.1-80 mg, 0.1-60 mg, 0.1-40 mg, 0.1-30 mg, 0.1-20 mg, 0.1-15 mg, 0.1-10 mg, 0.1-5 mg, 0.2-40 mg, 0.2-30 mg, 0.2-20 mg, 0.2-15 mg, 0.2-10 mg, 0.2-5 mg, 0.4-40 mg, 0.4-30 mg, 0.4-20 mg, 0.4-15 mg, 0.4-10 mg, 0.4-5 mg, 0.4-4 mg, 1-20 mg, 1-15 mg, or 1-10 mg. Typically, corticosteroids such as glucocorticoids are administered to the average adult human subject in doses of approximately 0.4 to 20 mg, for example, approximately 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0232] In some embodiments, the corticosteroid is typically administered to an average adult subject weighing approximately 70-75 kg in doses such as, for example, 0.001 or approximately 0.001 mg / kg (of the subject), 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.035 mg / kg, 0.04 mg / kg, 0.045 mg / kg, 0.05 mg / kg, 0.055 mg / kg, 0.06 mg / kg, 0.065 mg / kg, 0.07 mg / kg, 0.075 mg / kg. g, 0.08mg / kg, 0.085mg / kg, 0.09mg / kg, 0.095mg / kg, 0.1mg / kg, 0.15mg / kg, 0.2mg / kg, 0.25mg / kg, 0.30mg / kg, 0.35mg / kg, 0.40mg / kg, 0.45mg / kg, 0.50mg / kg, 0.55mg / kg, 0.60mg / kg, 0.65mg / It can be administered in doses of kg, 0.70 mg / kg, 0.75 mg / kg, 0.80 mg / kg, 0.85 mg / kg, 0.90 mg / kg, 0.95 mg / kg, 1 mg / kg, 1.05 mg / kg, 1.1 mg / kg, 1.15 mg / kg, 1.20 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.35 mg / kg, or 1.4 mg / kg.

[0233] Generally, the dose of corticosteroid administered depends on the specific corticosteroid, as there are differences in potency between different corticosteroids. Typically, it is understood that because drugs have different potencies, the doses required to achieve equivalent effects may differ. Equivalent doses in terms of potency for various glucocorticoids and routes of administration are well known. Information on equivalent steroid doses (non-chronotherapy) can be found in the British National Formulary (BNF) 37, March 1999.

[0234] In some embodiments, adverse events / reactions may be selected from one or more of the following: TIFF2026508791000009.tif225158

[0235] Other adverse reactions include gastrointestinal disorders: dry mouth; infection and parasitic disorders: fungal infections; metabolic and nutritional disorders: dehydration; nervous system disorders: ataxia, seizures, increased intracranial pressure; respiratory, thoracic and mediastinal disorders: respiratory failure, pulmonary edema; skin and subcutaneous tissue disorders: rash; and vascular disorders: bleeding.

[0236] In one embodiment, symptoms of cytokine release syndrome include, but are not limited to, fever, rigidity, fatigue, loss of appetite, myalgia, arthralgia, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxia, tachycardia, hypotension, dilated pulse, early increase in cardiac output, delayed decrease in cardiac output, hallucinations, tremors, gait changes, seizures, and death. In one embodiment, a method for grading CRS is described in Neelapu et al., Nat Rev Clin Oncol. 15(1):47-62 (2018) and Lee, et al., Blood 2014;124:188-195. In one embodiment, neurotoxic / neurological events may be graded according to the method described in Lee, et al, Blood 2014;124:188-195.

[0237] In some embodiments, adverse events are managed with tocilizumab (or another anti-IL6 / IL6R agent / antagonist) for toxicity prevention, corticosteroid therapy, or anticonvulsants. In some embodiments, adverse events are managed with one or more agents selected from GM-CSF, CSF1, GM-CSFR, or CSF1R inhibitors, anti-thymocyte globulins, renzilumab, maprilumab, cytokines, and anti-inflammatory agents.

[0238] In some embodiments, the Disclosure provides methods for preventing the occurrence of adverse reactions to the T-cell therapy of the Disclosure or for reducing the severity of adverse reactions. In some embodiments, the cell therapy is administered with one or more agents that prevent, delay the onset of, reduce the symptoms of, or treat adverse events, including cytokine release syndrome and neurotoxicity. In one embodiment, the agents are described above. In other embodiments, the agents are described below. In some embodiments, the agents are administered before, after, or concurrently with the administration of cells by one of the methods and dosages described elsewhere in this Spec. In one embodiment, the agents are administered to subjects who may be prone to disease but have not yet been diagnosed with the disease.

[0239] In this regard, the disclosed methods may include the administration of a “prophylactic effective dose” of tocilizumab, corticosteroid therapy, and / or anticonvulsants for toxicity prevention. In some embodiments, the methods include the administration of inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, rendilumab, mabrilimumab, cytokines, and / or anti-inflammatory agents. The pharmacological and / or physiological effects may be prophylactic, i.e., the effect completely or partially prevents the disease or its symptoms. “Prophylactic effective dose” may refer to an effective dose to achieve the desired prophylactic outcome (e.g., prevention of the onset of adverse reactions) in the required dose and duration.

[0240] In some embodiments, the method includes the management of adverse reactions in any subject. In some embodiments, adverse reactions are selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, serious infections, cytopenia, and hypogammaglobulinemia. In some embodiments, signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia / anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are identified and selected based on one or more biomarkers of adverse events.

[0241] In some embodiments, the method includes preventing CRS in chimeric receptor therapy or reducing its severity. In some embodiments, the engineered CAR T cells are inactivated after administration to the patient. In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. Patients exhibiting Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid therapy, or hypoxia requiring oxygen supplementation) should be monitored with continuous telecardiogram monitoring and pulse oximetry. In some embodiments, for patients exhibiting severe CRS, consider performing echocardiography to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered. In some embodiments, the method includes monitoring the patient daily for at least 7 days after infusion at a certified medical facility for signs and symptoms of CRS. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention at a hospital whenever signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as an indication for the first signs of CRS.

[0242] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients undergoing Grade 2 or higher neurotoxicity should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

[0243] In some embodiments, cell therapy is administered before, during / concurrently with, and / or after the administration (e.g., steroids) or treatment (e.g., dose reduction) of one or more agents that treat or prevent (are prophylactic) one or more symptoms of an adverse event. “Prophylactic effective dose” refers to the effective dose required to achieve the desired prophylactic outcome in the required dose and duration. In one embodiment, the prophylactic effective dose is used in the subject prior to or at an early stage of the disease. In one embodiment, the prophylactic effective dose would be less than the therapeutic effective dose. In one embodiment, the treatment or prevention of an adverse event is administered to any patient who is, is, or has previously received cell therapy. In some embodiments, a method for managing adverse events includes monitoring the patient daily for at least seven days after infusion at an accredited medical facility for signs and symptoms of neurotoxicity. In some embodiments, a method includes monitoring the patient for signs or symptoms of neurotoxicity and / or CRS for four weeks after infusion.

[0244] In some embodiments, the Disclosure provides two methods for managing adverse events in subjects receiving CAR T-cell therapy with steroids and anti-IL6 / anti-IL-6R antibodies. In one embodiment, the Disclosure provides a method of adverse event management in which corticosteroid therapy is initiated for the management of all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 1 neurological events. In one embodiment, the Disclosure provides a method for initiating tocilizumab for the management of all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 2 neurological events. In one embodiment, the Disclosure provides a method for reducing total steroid exposure in patients who have received adverse event management after CAR T-cell administration, the method comprising initiating corticosteroid therapy for the management of all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 1 neurological events, and / or initiating tocilizumab for all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 2 neurological events. In one embodiment, corticosteroids and tocilizumab are administered in a regimen selected from those exemplified in the Examples section. In one embodiment, the disclosure provides that early steroid use is not associated with an increased risk of severe infection, decreased CAR T cell proliferation, or reduced tumor response.

[0245] In one embodiment, this disclosure supports the safety of levetiracetam prophylaxis in CAR T-cell cancer therapy. In one embodiment, the cancer is NHL. In another embodiment, the cancer is R / R LBCL and the patient receives KTE-X19. Thus, in one embodiment, this disclosure provides a method for managing adverse events in a patient treated with CAR T cells, comprising administering a prophylactic dose of an anticonvulsant to the patient. In some embodiments, the patient is administered levetiracetam (e.g., 750 mg orally or intravenously twice daily) starting on day 0 of CAR T-cell therapy (post-pre-treatment) and if a neurological event occurs after discontinuation of prophylactic levetiracetam, or if a grade ≥ 2 neurotoxicity develops. In one embodiment, if the patient does not experience any grade ≥ 2 neurotoxicity, levetiracetam is reduced and discontinued as clinically indicated. In one embodiment, levetiracetam prophylaxis is combined with any other adverse event management protocol.

[0246] In one embodiment, the patient may be administered levetiracetam (750 mg orally or intravenously twice daily) starting on day 0. Upon the onset of a grade 2 or higher neurological event, the dose of levetiracetam is increased to 1000 mg twice daily. If the patient does not exhibit any grade 2 or higher neurological events, levetiracetam is gradually tapered and discontinued as clinically necessary. On day 2, the patient is also administered tocilizumab (8 mg / kg IV over 1 hour [not exceeding 800 mg]). Further tocilizumab (± corticosteroids) may be recommended upon the onset of grade 2 CRS in patients with comorbidities or in elderly patients, or otherwise in cases of grade 3 or higher CRS. Tocilizumab is initiated in patients exhibiting a Grade 2 or higher neurological event. Corticosteroids are added in patients with comorbidities, elderly patients, or in patients experiencing a Grade 3 or higher neurological event that worsens despite tocilizumab use.

[0247] In one embodiment, the disclosure provides that prophylactic steroid use appears to reduce the rate of severe CRS and NE to a similar extent as initial steroid administration. Accordingly, the disclosure provides a method for managing adverse events in CAR T-cell therapy, in which the patient is administered dexamethasone 10 mg PO on day 0 (pre-infusion), day 1, and day 2. Steroids may be initiated in cases of grade 1 NE and grade 1 CRS when no improvement is observed after 3 days of supportive care. Tocilizumab may be administered in cases of grade ≥ 1 CRS when no improvement is observed after 24 hours of supportive care. In one embodiment, the disclosure presents that management of adverse events in CAR T-cell therapy with an antibody that neutralizes and / or depletes GM-CSF prevents or reduces treatment-related CRS and / or NE in treated patients. In one embodiment, the antibody is rengilumab.

[0248] In some embodiments, adverse events are controlled by administering a drug / multiple drugs that are antagonists or inhibitors of IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the drug is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the drug is or comprises the anti-IL-6R antibody tocilizumab (atlizumab) or sarilumab. In some embodiments, the drug is the anti-IL-6R antibody described in U.S. Patent No. 8,562,991. In some examples, IL-6 targeting agents are anti-IL-6 antibodies, such as siltuximab, elcirimomab, ALD518 / BMS-945429, sirukmab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), and combinations thereof. In some embodiments, the agent may neutralize IL-6 activity by inhibiting ligand-receptor interaction. In some embodiments, the IL-6 / IL-6R antagonist or inhibitor is IL-6 mutein, for example, as described in U.S. Patent No. 5591827. In some embodiments, the agent that is an IL-6 / IL-6R antagonist or inhibitor is a small molecule, protein or peptide, or nucleic acid.

[0249] In some embodiments, other agents that may be used to manage adverse reactions and their symptoms include cytokine receptors or cytokine antagonists or inhibitors. In some embodiments, the cytokine or receptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R / CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFα, TNFR1, for example, TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), IL-1, and IL-1Rα / IL-1β. In some embodiments, the drug includes siltuximab, sarilumab, olokizumab (CDP6038), elcilimomab, ALD518 / BMS-945429, silkumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the drug is a cytokine antagonist or inhibitor, such as transforming growth factor β (TGFβ), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNFα, IL-1, interferon γ (IFN-γ), or monocyte chemotactic protein-1 (MCP-1). In some embodiments, the drug targets cytokine receptors (e.g., inhibits or is an antagonist thereof), such as the TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), and combinations thereof.In some embodiments, the drug is administered before, after, or concurrently with the administration of cells by one of the methods and dosages described elsewhere in this specification.

[0250] In some embodiments, the drug is administered in doses of 1 mg / kg to 10 mg / kg or about 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg or about 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg or about 2 mg / kg to 6 mg / kg, 2 mg / kg to 4 mg / kg or about 2 mg / kg to 4 mg / kg, or 6 mg / kg to 8 mg / kg or about 6 mg / kg to 8 mg / kg (including both ends of the range), or the drug is administered in doses of at least 2 mg / kg or at least about 2 mg / kg or about 2 mg / kg, at least 4 mg / kg or at least about 4 mg / kg or about 4 mg / kg, at least 6 mg / kg or at least about 6 mg / kg or about 6 mg / kg, or at least 8 mg / kg or at least about 8 mg / kg or about 8 mg / kg. In some embodiments, the drug is administered in doses of approximately 1 mg / kg to 12 mg / kg, for example, 10 mg / kg or approximately 10 mg / kg. In some embodiments, the drug is administered by intravenous infusion. In one embodiment, the drug is tocilizumab. In some embodiments, the drug, for example, tocilizumab in particular, is administered before, after, or concurrently with cell administration by one of the methods and doses described elsewhere in this specification.

[0251] In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. If CRS is observed or suspected, it may be managed according to the recommendations of Protocol A, and this may be used in combination with other treatments of the Disclosure, including neutralization or reduction of the CSF / CSFR1 system. Patients exhibiting Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid resuscitation, or hypoxia requiring oxygen supplementation) should be monitored with continuous telecardiogram monitoring and pulse oximetry. In some embodiments, for patients exhibiting severe CRS, consider performing echocardiography to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered. In some embodiments, a biosimilar or equivalent of tocilizumab may be used instead of tocilizumab in the methods disclosed herein. In other embodiments, another anti-IL6R may be used instead of tocilizumab.

[0252] In some embodiments, adverse events are managed according to the following protocol (Protocol A). TIFF2026508791000010.tif130170(a)Lee DW et al.(2014).Current concepts in the diagnosis and management of cytokine release syndrome.Blood.2014 Jul 10;124(2):188-195. (b) Refer to Protocol B for the management of neurotoxicity. (c) For further details, see ACEMTRA® (tocilizumab) prescribing information, https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). The initial US approval was indicated in 2010.

[0253] Neurotoxicity In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients undergoing Grade 2 or higher neurotoxicity should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. Consider non-sedating antiseizure agents (e.g., levetiracetam) for seizure prevention in any Grade 2 or higher neurotoxicity. The following treatments may be used in combination with other treatments of the Disclosure, such as neutralization or reduction of the CSF / CSFR1 axis.

[0254] In some embodiments, adverse events are managed according to the following protocol (Protocol B). TIFF2026508791000011.tif97170

[0255] Further safety management methods using corticosteroids The administration of corticosteroids and / or tocilizumab in Grade 1 can be considered prophylactic. Supportive care may be provided in all protocols for all CRS and NE severity grades. In one embodiment of a protocol for managing adverse events associated with CRS, tocilizumab and / or corticosteroids, the following is administered: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only in cases with comorbidities or advanced age); and / or corticosteroids (only in cases with comorbidities or advanced age); Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids. In another embodiment of the protocol for managing adverse events associated with CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: tocilizumab (if no improvement after 3 days); and / or corticosteroids (if no improvement after 3 days); Grade 2 CRS: tocilizumab; and / or corticosteroids; Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids, high dose.

[0256] In one embodiment of the protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following is administered: Grade 1 NE: no tocilizumab; no corticosteroids; Grade 2 NE: no tocilizumab; no corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids (standard dose only if there is no improvement with tocilizumab); Grade 4 NE: tocilizumab; and / or corticosteroids. In another embodiment of the protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following is administered: Grade 1 NE: no tocilizumab; and / or corticosteroids; Grade 2 NE: tocilizumab; and / or corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids, high dose; Grade 4 NE: tocilizumab; and / or corticosteroids, high dose. In one embodiment, corticosteroid therapy is initiated at grade 2 or higher CRS, and tocilizumab is initiated at grade 2 or higher CRS. In another embodiment, corticosteroid therapy is initiated at grade 1 or higher CRS, and tocilizumab is initiated at grade 1 or higher CRS. In another embodiment, corticosteroid therapy is initiated at grade 3 or higher NE, and tocilizumab is initiated at grade 3 or higher CRS. In yet another embodiment, corticosteroid therapy is initiated at grade 1 or higher CRS, and tocilizumab is initiated at grade 2 or higher CRS. In some embodiments, prophylactic use of tocilizumab administered on day 2 may reduce the percentage of grade 3 or higher CRS. One or more corticosteroids may be administered in any dose and frequency that can be appropriate to the severity / grade of adverse events (e.g., CRS and NE). In yet another embodiment, corticosteroid administration includes oral or IV administration of dexamethasone 10 mg 1 to 4 times per day. Another embodiment, sometimes referred to as “high-dose” corticosteroids, involves intravenous administration of methylprednisone 1 g / day alone or in combination with dexamethasone. In some embodiments, one or more corticosteroids are administered at a dose of 1 to 2 mg / kg per day.Generally, the dose of corticosteroid administered depends on the specific corticosteroid, as there are differences in potency between different corticosteroids. Typically, it is understood that because drugs have different potencies, the doses required to achieve equivalent effects may differ. Equivalents in terms of potency for various glucocorticoids and routes of administration are well known. Information on equivalent steroid dosing (in a non-chronotherapy manner) can be found in the British National Medicines Collection (BNF) 37, March 1999. This application also provides doses and administrations of cells prepared by the method of this application, for example, an infusion bag for CD19-directed genetically modified autologous T-cell immunotherapy contains approximately 68 mL of chimeric antigen receptor (CAR)-positive T-cell suspension for infusion. In some embodiments, CAR T-cells are formulated in approximately 40 mL for infusion. In some embodiments, the CAR T cell product is formulated in a total volume of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900, and 1000 mL. In one embodiment, the dose and administration of cells prepared by the method of this application, for example, the infusion bag of CD19-directed genetically modified autologous T cell immunotherapy, is 1 × 10 in about 40 mL. 6 Contains a suspension of CAR-T positive cells. The target dose is approximately 1 × 10⁶ cells per kg of body weight. 6 ~about 2×10 6 These may be CAR-positive surviving T cells, up to 2 × 10⁶ 8 These could be CAR-positive surviving T cells.

[0257] In some embodiments, the dosage form comprises a cell suspension for infusion in a single-dose patient-specific infusion bag, the route of administration is intravenous, and the entire contents of each single-dose patient-specific bag are infused over 30 minutes by gravity or a peristaltic pump. In one embodiment, the administration regimen is 2.0 × 10 6 Individual anti-CD19 CAR T cells / kg body weight (±20%), maximum dose 2 × 10⁶ 8 This is a single infusion consisting of 100 anti-CD19 CAR T cells (for a subject weighing ≥100 kg). In some embodiments, the T cells constituting the dose are CD19 CAR-T cells.

[0258] In some embodiments, the CD19-directed T-cell immunotherapy is KTE-X19, which is prepared as described elsewhere in this application. In one embodiment, KTE-X19 may be used to treat MCL, ALL, CLL, SLL, and any other B-cell malignancies. In some embodiments, the CD19-directed genetically modified autologous T-cell immunotherapy is Axi-cel® (YESCARTA®, axicaptagensilolucel), which is prepared by one of the methods of this application. The amount of CAR T cells, dosing regimens, administration methods, subjects, and cancers within the scope of these methods are described elsewhere in this application, either alone or in combination with other chemotherapeutic agents, with or without pretreatment, for any of the patients described elsewhere in this application.

[0259] The following embodiments are intended to illustrate various aspects of this application. Therefore, any particular embodiment considered should not be construed as a limitation on the scope of this application. For example, the following embodiments involve T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), but those skilled in the art will understand that the methods described herein may be applied to immune cells transduced with any CAR. Those skilled in the art will understand that various equivalents, changes, and modifications can be made without departing from the scope of this application, and that such equivalent embodiments are included herein. Furthermore, all references cited herein are incorporated herein by reference in their entirety, as fully described herein.

[0260] The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, dictionaries, documents, manuscripts, genome database sequences, and scientific literature cited herein are incorporated herein by reference.

[0261] Other features and advantages of this disclosure will become apparent from the examples. [Examples]

[0262] Example 1 This example describes a phase 2 multicenter trial (ZUMA-2) evaluating the efficacy of brexucabtagene autoleucel (KTE-X19) in patients with relapsed / refractory mantle cell lymphoma (R / R MCL) who have not been previously treated with Bruton's tyrosine kinase inhibitors (BTKi).

[0263] This Phase 2 trial is a multicenter, open-label study evaluating the efficacy of Brexu-cel in patients with recurrent / recurrent MCL. Key eligibility criteria for Cohort 3 (NCT04880434) include patients aged 18 years or older with MCL who have received 1 to 5 prior regimens including anthracycline, bendamustine, or high-dose cytarabine-containing chemotherapy and an anti-CD20 monoclonal antibody, but have not received prior BTKi therapy. A prior history of allogeneic stem cell transplantation (alloSCT) is acceptable if donor cells are not detected by chimeric phenomenon more than 100 days after allogeneic stem cell transplantation. Patients will receive fludarabine 30 mg / m² administered on days -5, -4, and -3. 2 / day and cyclophosphamide 500mg / m² 2 After receiving pre-treatment chemotherapy on day / , followed by 2 x 10 on day 0. 6 Patients receive a single infusion of brexu-cel at a targeted dose of anti-CD19 CAR T cells / kg. At the discretion of the principal investigator, bridging therapy with dexamethasone, radiotherapy, specific chemotherapy, or any combination thereof is recommended for all patients in Cohort 3, especially those with rapidly progressing disease, clinical exacerbation, or high disease burden at screening.

[0264] The primary endpoint is the Objective Level Reaction (ORR), assessed by an independent Institutional Review Board (IRB) according to the Lugano classification. Secondary endpoints include safety, duration of response, progression-free survival, overall survival, circulating CAR T cell and cytokine levels, and changes in patient-reported outcomes over time. In the pivot cohort, the EuroQol 5-dimensional (EQ-5D) score was assessed only up to 6 months, whereas for Cohort 3, both the EQ-5D and the European Organisation for Research and Treatment of Cancer (EORTC-QLQ-C30) Quality of Life Questionnaire score were assessed over time. Minimally residual disease was assessed for Cohort 3 up to 24 months by next-generation sequencing of ctDNA.

[0265] Approximately 90 patients will be enrolled in Cohort 3, with a target ORR of 75%. It is hypothesized that the observed ORR will significantly exceed the historical control rate of 57% based on systematic literature review and meta-analysis. The primary analysis of Cohort 3 will be conducted after 86 patients have been enrolled, treated with Brexu-cel, and have had the opportunity to evaluate their response at 6 months after the first objective response or 9 months after Brexu-cel infusion, whichever comes first. ZUMA-2 Cohort 3 is currently enrolling patients at 41 sites in the United States, France, Germany, the Netherlands, Spain, and the United Kingdom.

[0266] Example 2 This example describes the evaluation of persistent response after brexcabutadiene autolucel (KTE-X19) in the ZUMA-2 study for relapsed / refractory mantle cell lymphoma (R / R MCL). The study design is shown in Figure 1. After 35.6 months of follow-up in ZUMA-2, brexu-cel showed an objective response rate (ORR; complete response [CR] + partial response [PR]) of 91% (95% CI, 81.8–96.7), a CR rate of 68% (95% CI, 55.2–78.5), a median duration of response (DOR) of 28.2 months (95% CI, 13.5–47.1), and a median overall survival (OS) of 46.6 months (95% CI, 24.9, not estimable), which was not achieved in all 68 treated patients with CR. To identify factors associated with the long-term response to KTE-X19, patient and product characteristics were evaluated based on the response status at 24 months post-infusion in ZUMA-2.

[0267] Methods: Key ZUMA-2 eligibility criteria include: receiving 1-5 preceding regimens containing anthracycline or bendamustine-containing chemotherapy, anti-CD20 monoclonal antibody, and BTKi, followed by leukocytosis and pre-treatment chemotherapy, and then brexu-cel (2 × 10⁻¹⁰ 6Adults (18 years or older) with R / R MCL who received a single infusion of anti-CD19 CAR T cells / kg. The primary endpoint was the objective response rate (ORR), referring to complete response (CR) + partial response (PR). This was evaluated by an independent radiological review committee (IRRC) according to the Lugano classification. Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and adverse events (AEs). Post-hoc evaluations of patients, disease, pharmacokinetics, and product characteristics were reported based on the response status (continuation vs. relapse) at 24 months. Baseline patient and disease characteristics, subsequent treatments, product characteristics, and pharmacological outcomes were evaluated based on the response status at 24 months after brexu-cel infusion: Continuing responders: patients with continued response at the 24-month evaluation; Relapsed responders: patients who relapsed before the 24-month evaluation but still responded; Non-responders: patients without a response. DOR was evaluated in patients with continued response and those with relapsed response. Statistical analysis: The time-to-event endpoint was analyzed using the Kaplan-Meier method. All subgroup analyses were descriptive.

[0268] Results: At a median follow-up of 35.6 months (range, 25.9–56.3), 74 patients were enrolled, underwent leukocytosis, and 68 patients received brexu-cel (Figure 2). TIFF2026508791000012.tif166154 a Bridging therapy was administered after leukocytosis and before pre-treatment chemotherapy with ZUMA-2. Compared to relapsed responders who received bridging therapy, the proportion of continuing responders was lower, the Eastern Cooperative Oncology Group Performance Status (ECOG PS) score was 1, and the median baseline tumor burden (sum of diameter products) was approximately four times smaller in continuing responders compared to relapsed responders (Table 1). The median number of prior therapies was 3 in both subgroups, and the proportion of continuing responders was lower compared to relapsed responders who received prior platinum therapy (Table 1). Ibrutinib was more commonly the last prior therapy in patients with continued responses versus those with relapsed responses, although a similar proportion received acalabrutinib as their last prior therapy (Table 2). The median time from last prior therapy to brexu-cel infusion was similar between patients with continued responses and those with relapsed responses, but more than twice as long in patients without responses, although the smaller sample size may have contributed to this difference. Sixty-two patients achieved complete response (CR) or partial response, while three were excluded from this analysis for not reaching the 24-month evaluation visit. Of the 59 evaluable patients who responded, 29 (47%) had continued response at 24 months (continued responses), and 30 (48%) had relapsed prior to 24 months (relapsed responses). Six patients did not respond (non-responses). At baseline, the median age was 65 years, and the median number of prior therapies was 3 in both subgroups. Among continued responses versus relapsed responses, 66% had ibrutinib versus 43%, and 14% had acalabrutinib as their last prior therapy, with a median (range) time from last prior therapy being 63 months (26–748) versus 64.5 months (22–443). Compared to patients who experienced a relapsed response, a smaller proportion of patients who experienced a continued response received bridging therapy (53% vs. 21%, respectively) and prior platinum therapy (40% vs. 10%). Conversely, similar proportions received prior bendamustine therapy (53% vs. 45%), prior proteosome inhibitor therapy (37% and 41%), and prior autologous stem cell transplantation (37% vs. 48%).

[0269] At baseline, a larger proportion of patients with continued responses had an ECOG score of 0 compared to patients with relapsed responses (79% vs. 57%, respectively), and the median (range) tumor burden (SPD) was 935.1 (260–6133) in patients with continued responses and 4233.6 (386–14390) in patients with relapsed responses. The incidence of high-risk features was similar between patients with continued and relapsed responses, with 66% and 60% having a baseline Ki-67 proliferation index score of ≥30%, 10% and 10% having TP53 mutations, 45% and 37% having elevated lactose dehydrogenase levels (≥ULN to ≤1.5ULN), and 10% and 13% having a high-risk International Prognostic Index score for mantle cell lymphoma (>6), respectively. TIFF2026508791000014.tif237170

[0270] In continuing response patients with CR (n=28), the median (range) DOR was not reached (46.7-unestimable), while in relapsed response patients with CR (n=15, Table 3), it was 8.3 months (5-13.6). The median time to initial response for continuing response patients versus relapsed response patients was 1 month (range, 0.9-3.1; n=29) versus 1 month (range, 0.8-1.7; n=30). The median time to complete response for continuing response patients versus relapsed response patients was 3 months (range, 0.9-35.1; n=28) versus 3 months (range, 0.8-9.0; n=15). The median time to conversion from SD or PR to CR for continuing response patients versus relapsed response patients was 2.3 months (range, 1.8-34.1; n=16) versus 2.4 months (range, 2.0-8.1; n=8).

[0271] The median (95% CI) DOR in continuing response patients with high baseline LDH concentrations (n=12) was 47.1 months (24.8-not estimable), and in relapsed response patients with high baseline LDH concentrations (n=5) it was 8.3 months (4.7-NE). TIFF2026508791000015.tif229170TIFF2026508791000016.tif233170TIFF2026508791000017.tif104167 Of those who responded to relapse, 67% received subsequent chemotherapy due to data cutoff, the most common of which were radiotherapy (23%), dexamethasone (23%), rituximab (23%), venetoclax (20%), and lenalidomide (20%); patients were able to receive multiple and multiple lines of follow-up therapy. TIFF2026508791000018.tif66170 Median [range] Peak (102.4 [0.3-2241.6] vs. 59.9 [1.6-2589.5]) and Area under the curve (1487 [3.8-.0002] vs. 688.2 [19-.0003]) CAR T cell levels were approximately twice as high in continuing responders compared to relapsed responders (Table 7). A moderate increase in the median [range] of the total number of injected CCR7+ cells was observed in continuing responders versus relapsed responders (119.8 [37-249.9] vs. 89.1 [6.1-353.4]), suggesting the need for further investigation into the role of continuous memory T cell differentiation in achieving persistent response. The characteristics of the TIFF2026508791000019.tif30170 product were nearly identical between continuing and relapsed responders, and were accompanied by a moderate increase in the median total number of injected CCR7+ T cells observed in continuing responders versus relapsed responders (Table 8). TIFF2026508791000020.tif252170TIFF2026508791000021.tif44170TIFF2026508791000 022.tif251170TIFF2026508791000023.tif44170TIFF2026508791000024.tif252170TIFF2 026508791000025.tif44170TIFF2026508791000026.tif252170TIFF2026508791000027.tif44170 Peripheral blood T cells from relapsed and non-response patients show a more pronounced CD8+CD27-CD28+ effector memory phenotype compared to patients with continued response. Continued responders are enriched with peripheral CD4 T cells that maintain immature CD27+ expression and activated CD8 effector memory T cells.

[0272] At a median follow-up of approximately 3 years, brexu-cel continued to demonstrate persistent responses, with 47% of responders still maintaining a response 24 months after infusion. Persistent responses were observed in patients with high-risk disease characteristics, suggesting that brexu-cel may have the potential to deliver persistent responses in patients with R / R MCL, which typically has a poor prognosis. Ibrutinib was more generally the last prior therapy in continuing responders versus relapsed responders. In summary, continuing responders had lower ECOG PS scores, lower tumor burden, less frequent use of prior platinum therapy or bridging therapy, and less intense regimens for previous relapses compared to relapsed responders, suggesting the potential for greater benefit from brexu-cel when administered at an earlier stage of disease progression. Median peak and AUC CAR T cell levels were approximately twice as high in continuing responders than in relapsed responders, suggesting that the degree of CAR T cell proliferation may predict the persistence of the response. The moderate increase in the median total number of injected CCR7+ cells and the maintenance of CD27+ peripheral T cells observed in patients with sustained responses versus those with relapsed responses may suggest a potential role of sequential memory T cell differentiation in achieving lasting responses.

[0273] Example 3 This example describes a phase 2 open-label, multicenter basket study (ZUMA-25) evaluating the safety and efficacy of brexkabutadiene autolucel in adults with rare B-cell malignancies, including Waldenström macroglobulinemia, Richter transformation, Burkitt lymphoma, and hairy cell leukemia.

[0274] The primary objective of this study is to evaluate the efficacy of brexkabutadiene autolucel in four rare B-cell malignancies. This study will utilize a basket study design with separate indication-specific sub-studies to examine relapsed / refractory Waldenström macroglobulinemia (r / rWM), relapsed / refractory Richter transformation (r / rRT), relapsed / refractory Burkitt lymphoma (r / rBL), and relapsed / refractory hairy cell leukemia (r / rHCL).

[0275] Sub-study A: The primary objective of this sub-study is to evaluate the efficacy of brexcabutadiene autolucer in participants with r / r WM by determining the combined rate of complete response (CR) and very good partial response (VGPR) by central assessment. Participants will receive lymphocyte depletion chemotherapy consisting of fludarabine 30 mg / m² / day and cyclophosphamide 500 mg / m² / day for three consecutive days from day -5 to day -3, followed by a two-day rest period (days -2 and -1), and then, in subjects weighing over 100 kg, a target dose of 2 × 10⁶ anti-CD19 chimeric antigen receptor (CAR) T cells / kg or 1 × 10⁶ anti-CD19 CAR T cells / kg, or 2 × 10⁶ 8 individual or 1 x 10 8 Individuals received a single infusion of brexkabutadiene-autolucel in a flat dose of anti-CD19 CAR T cells.

[0276] Sub-study B: The primary objective of this sub-study is to evaluate the efficacy of brexcabutadiene autolucer for diffuse large B-cell lymphoma-Richter transformation (DLBCL-RT) in participants undergoing r / r RT by determining the objective response rate (ORR) by central assessment. Participants received lymphocyte depletion chemotherapy with fludarabine 30 mg / m² / day and cyclophosphamide 500 mg / m² / day for three consecutive days from day -5 to day -3, followed by a two-day rest period (days -2 and -1), and then, in subjects weighing over 100 kg, a target dose of 2 × 10⁶ anti-CD19 CAR T cells / kg or 1 × 10⁶ anti-CD19 CAR T cells / kg, or 2 × 10⁶ 8 individual or 1 x 10 8 Individuals received a single infusion of brexkabutadiene-autolucel in a flat dose of anti-CD19 CAR T cells.

[0277] Sub-study C: The primary objective of this sub-study is to evaluate the efficacy of brexcabutadiene autolucel in participants with r / r BL by determining ORR by central assessment. Participants received lymphocyte depletion chemotherapy consisting of fludarabine 30 mg / m² / day and cyclophosphamide 500 mg / m² / day for three consecutive days from day -5 to day -3, followed by a two-day rest period (days -2 and -1), and then, in subjects weighing over 100 kg, a target dose of 2 × 10⁶ anti-CD19 CAR T cells / kg or 1 × 10⁶ anti-CD19 CAR T cells / kg, or 2 × 10⁶ 8 individual or 1 x 10 8 Individuals received a single infusion of brexkabutadiene-autolucel in a flat dose of anti-CD19 CAR T cells.

[0278] Sub-study D: The primary objective of this sub-study is to evaluate the efficacy of brexcabutadiene autolucel in participants with r / r HCL by determining ORR by central assessment. Participants received lymphocyte depletion chemotherapy consisting of fludarabine 30 mg / m² / day and cyclophosphamide 500 mg / m² / day for three consecutive days from day -5 to day -3, followed by a two-day rest period (days -2 and -1), and then, in subjects weighing over 100 kg, a target dose of 2 × 10⁶ anti-CD19 CAR T cells / kg or 1 × 10⁶ anti-CD19 CAR T cells / kg, or 2 × 10⁶ 8 individual or 1 x 10 8 Individuals received a single infusion of brexkabutadiene-autolucel in a flat dose of anti-CD19 CAR T cells.

[0279] The following specific comprehensive criteria apply to all indications: 1) Male or female aged 18 years or older at the time of signing informed consent; 2) Any prior therapy toxicity must be stable and reduced to Grade 1 or lower (excluding clinically insignificant toxicity such as alopecia); 3) Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; 4) Adequate hematological function as indicated by absolute neutrophil count (ANC) ≥ 500 / μL, platelet count ≥ 50,000 / μL, and hemoglobin level ≥ 8 g / dL (unless lower values ​​are due to an underlying disease); 5) absolute lymphocyte count ≥ 100 / μL; 6) Creatinine clearance (estimated by the Cockcroft-Gault formula) ≥ 60 mL / min, serum alanine aminotransferase and aspartate aminotransferase levels ≤ 2.5 × upper limit of normal (ULN), or ≤ 5 × ULN if there is demonstrated liver lesion, and total bilirubin level ≤ 1.5×ULN (excluding subjects with no evidence of pericardial exudation as determined by echocardiography (ECHO) or multi-gate acquisition scan (MUGA) and no clinically significant electrocardiogram (ECG) findings), no clinically significant pleural effusion, and sufficient renal, hepatic, pulmonary, and cardiac function as defined by baseline oxygen saturation >92% at room temperature;7) The following washout periods must be met before leukocytosis / registration: Corticosteroid therapy at pharmacological doses (prednisone ≥ 5 mg / day or equivalent doses of other corticosteroids) must be avoided for 7 days prior to leukocytosis, BTK inhibitors (e.g., ibrutinib or acalabrutinib), and unless otherwise specified in the subprotocol, must be avoided for at least 1 week or 5 half-lives prior to leukocytosis. Furthermore, any antineoplastic drugs used in previous therapies must be avoided within one week or five half-lives prior to leukocyte apheresis, whichever is shorter; systemic inhibitory / stimulative immune checkpoint molecular therapies (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists) must be avoided for at least three half-lives prior to leukocyte apheresis; alemtuzumab must be avoided at least six months prior to enrollment; PEG-asparaginase must be avoided at least three weeks prior to enrollment; cladribine and pentostatin must be avoided three months prior to enrollment; donor lymphocyte infusion within 28 days prior to enrollment; and any treatment with immunosuppressive antibodies used within four weeks prior to enrollment (e.g., anti-CD20, anti-tumor necrosis factor [TNF], anti-interleukin [IL]6, or anti-IL6 receptor). However, this does not apply if this treatment is included in a pre-treatment regimen or bridging regimen, in which case a 7-day washout period is required before leukocyte apheresis; and 8) women of childbearing potential must have negative serum or urine pregnancy tests (women who have been sterilized or who have been postmenopausal for at least two years are not considered of childbearing potential).

[0280] The following specific exclusion criteria are common to all indications: 1) Prior CAR therapy or other genetically modified T-cell therapy; 2) Prior treatment with any anti-CD19 therapy; 3) History of severe immediate-type hypersensitivity reaction to aminoglycosides; 4) History of severe immediate-type hypersensitivity reaction to cyclophosphamide or fludarabine; 5) Presence or suspicion of a history of uncontrolled fungal, bacterial, viral, or other infection requiring intravenous administration of antimicrobial agents for management. If responding to active treatment, simple urinary tract infections and simple bacterial pharyngitis are acceptable. Patients with simple urinary tract infections and uncomplicated bacterial pharyngitis who respond to active treatment are eligible only if they meet the criterion of being fever-free (i.e., having a temperature below 38°C) for at least 24 hours prior to the investigator confirming the patient's eligibility. 6) HIV-positive patients with an undetectable viral load by quantitative polymerase chain reaction (qPCR) and a CD4 count greater than 200 cells / uL unless they are taking appropriate anti-HIV medication; 7) Acute or chronic active hepatitis B or C infection. Subjects with a history of hepatitis infection must be cleared of infection as determined by standard serological and genetic testing in accordance with current Infectious Diseases Society of America guidelines or applicable national guidelines. 8) The presence of a dedicated central venous access catheter for any indwelling line or drain (e.g., percutaneous nephrostomy, indwelling Foley catheter, bile drainage duct, or pleural / peritoneal / pericardial catheter) is permitted; 9) A history or present illness of detectable cerebrospinal fluid (CSF) malignant cells or brain metastases, unless otherwise specified in the sub-study eligibility criteria; 10) A history or present illness of central nervous system (CNS) disorders (such as cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, or any autoimmune disease involving the CNS, posterior reversible encephalopathy syndrome, or cerebral edema with structural defects confirmed by appropriate imaging). A history of stroke or transient ischemic attack within 12 months prior to enrollment.Patients with paroxysmal disorders requiring active anticonvulsant medication; 11) presence of atrial or ventricular lymphoma; 12) history of myocardial infarction, cardiovascular angioplasty or stent placement, unstable angina, or other clinically significant cardiac disease within 12 months prior to registration; 13) need for emergency treatment due to tumor mass effects (e.g., vascular compression, intestinal obstruction, or transmural gastric lesion); 14) presence of primary immunodeficiency; 15) history of autoimmune disease (e.g., Crohn's disease, rheumatoid arthritis, systemic lupus) within the past two years that has resulted in end-organ damage or requires systemic immunosuppression / systemic disease modifiers. 16) A history of deep vein thrombosis or pulmonary embolism requiring therapeutic anticoagulation within 6 months prior to enrollment; 17) Any medical condition that may impair the evaluation of the safety or efficacy of the study treatment; 18) A history of a severe immediate-type allergic reaction to any of the drugs used in this study; 19) Prediction of the need for live vaccines within 6 weeks prior to the planned initiation of a lymphocyte apheresis chemotherapy regimen and for the first 12 months after brexkabutagen infusion; 20) Pregnant or lactating women (due to the potentially dangerous effects of preparatory chemotherapy on the fetus or infant). Women who have undergone sterilization or have been postmenopausal for at least 2 years were considered to have no potential for pregnancy; 21) No intention to plan for conception from the time of consent until 6 months after brexkabutagen autolucel infusion; 22) In the judgment of the principal investigator, the subject is unlikely to complete all study-specific visits or procedures, including follow-up visits, or to comply with the study requirements for participation.

[0281] Regarding Waldenström macroglobulinemia, the specific inclusion criteria for the sub-study include: a clinicopathological diagnosis of Waldenström macroglobulinemia; two or more prior treatments for WM (including BTKi and chemotherapy with disease progression or no response); a need for treatment according to guidelines; and a measurable disease (IgM levels greater than twice the upper limit of normal). Regarding Waldenström macroglobulinemia, the sub-study-specific exclusion criteria include: allogeneic SCT; self-SCT is acceptable if it has been more than 6 months; and a prior history of CNS infiltration (Bing-Neel syndrome) unless there are no lesions on brain MRI and CSF.

[0282] With regard to Richter transformation, the specific inclusion criteria for the sub-study include: a confirmed diagnosis of CLL based on the 2018 IWCLL criteria, and histologically confirmed Richter transformation to the DLBCL subtype; at least one measurable disease site based on the 2014 Lugano criteria; and R / R RT is defined as a single primary refractory disease or relapse after one or more lines of chemotherapy. With regard to Richter transformation, the sub-study-specific exclusion criteria include: less than 3 months prior to screening for a preceding allogeneic or autologous SCT and / or less than 4 months prior to the planned infusion of brexcabutadiene autolucel, and the presence of active graft-versus-host disease following a preceding stem cell transplant.

[0283] For Burkitt lymphoma / leukemia, sub-study-specific inclusion criteria include: histologically confirmed mature B-cell NHL Burkitt lymphoma / leukemia; R / R BL defined as a single primary refractory disease or relapsed after one or more lines of chemotherapy including anthracyclines; and measurable disease by radiological criteria or involvement of isolated bone marrow. For Burkitt lymphoma / leukemia, sub-study-specific exclusion criteria include patients with prior allogeneic stem cell transplantation < 3 months prior to screening, and patients with active graft-versus-host disease following prior allogeneic stem cell transplantation.

[0284] For hairy cell leukemia, the sub-study-specific inclusion criteria include histologically confirmed hairy cell leukemia; neutrophils < 1.0 × 10⁻⁶. 9 / L, platelets <100×10 9 / L, hemoglobin <11 g / dL, need for treatment based on symptomatic splenomegaly or lymphadenopathy; at least two prior systemic therapies including PNA and moxetumomab Pasdotox, if eligible and available.

[0285] In certain embodiments, bridging therapy may be administered after leukocyte apheresis and before lymphocyte depletion preconditioning chemotherapy. In certain further embodiments, bridging therapy must be completed at least 7 days before lymphocyte depletion preconditioning chemotherapy or 5 days before its half-life.

[0286] At the discretion of the healthcare provider, patients with Richter transformation may receive bridging therapy selected from the group consisting of rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine, and prednisone (R-CHOP); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R); Bruton's tyrosine kinase inhibitors (BTKi) (BTKi) ± VTX-2337; dexamethasone; and irradiation. Bridging therapy regimens for patients with Richter transformation include those outlined in Table 10. The listed doses are merely examples and may be adjusted according to age, comorbidities, or regional or institutional guidelines. TIFF2026508791000028.tif156170 Abbreviations: AUC, Area under the curve; BID, Twice daily; BTK, Bruton's tyrosine kinase; DA-EPOCH-R, Dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab, IV, Intravenous; PO, Oral; R-CHOP, Rituximab containing cyclophosphamide, doxorubicin, vincristine, and prednisolone; R-GEMOX, Rituximab, gemcitabine, and oxaliplatin; R-ICE, Rituximab, ifosfamide, carboplatin, and etoposide

[0287] At the discretion of the healthcare provider, patients with Burkitt lymphoma may receive bridging therapy selected from the following groups: rituximab, ifosfamide, carboplatin, and etoposide (R-ICE); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R); rituximab, gemcitabine, and oxaliplatin (R-GEMOX); cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride, and dexamethasone (HyperCVAD); dexamethasone; and irradiation.

[0288] Bridging therapy regimens for patients with Burkitt lymphoma include those outlined in Table 11. The listed doses are merely examples and may be adjusted according to age, comorbidities, or regional or institutional guidelines. TIFF2026508791000029.tif177170 Abbreviations: BID, twice daily; CrCl, creatinine clearance; DA-EPOCH-R, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab; hyper-CVAD, hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone; IV, intravenous; PO, oral; R-GEMOX, rituximab containing gemcitabine and oxaliplatin; R-ICE, rituximab containing ifosfamide, carboplatin, and etoposide.

[0289] At the discretion of the healthcare provider, patients with Waldenström macroglobulinemia may receive ibrutinib bridging therapy.

[0290] Example 4 Brexukabutadiene autolucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for relapsed / refractory mantle cell lymphoma (R / R MCL). In the critical Phase 2 ZUMA-2 trial, after 3-year follow-up, 91% of MCL patients progressing on BTK inhibitors responded to brexu-cel therapy, with a median duration of response (DOR) of 28.2 months. This case study presents data evaluating the patient, product, and PK characteristics of ZUMA-2 in patients who responded to continued responses (patients who responded at 24-month evaluation), relapsed responses (patients who initially responded but relapsed before 24-month evaluation), and non-responders.

[0291] At a median follow-up of 35.6 months, 68 patients received brexu-cel, with 91% (n=62) achieving a response (CR or PR) and 9% (n=6) not responding. Of the responders, 29 had continued response at the 24-month evaluation (28 CRs and 1 PR) (15 CRs and 14 PRs). Continued responders had lower baseline median tumor burden (total product diameter, 935 vs. 4861 mm) compared to relapsed responders. 2 ) and had a higher frequency of Eastern Cooperative Oncology Group (ECOG) performance status 0 (79% vs. 59%). Compared to patients who experienced a relapsed response, the proportion of patients who received prior platinum therapy (10% vs. 41%) or bridging therapy (21% vs. 52%) was lower.

[0292] The median duration of response (DOR) was 47.1 months (95% CI, 36.5 months undeterminable) and 5.0 months (95% CI, 2.2–8.3) in patients with continued response and those with relapsed response, respectively. The median DOR was similar regardless of response status between patients with high baseline lactate dehydrogenase levels and those with low baseline LDH levels.

[0293] The peak and median area under the curve for CAR T cell proliferation were approximately twice as large in relapsed responders compared to continuing responders. Product characteristics were nearly identical between continuing and relapsed responders, with a moderate increase in the median total number of naive-like injected chemokine receptor 7 (CCR7)-positive T cells observed in both groups.

[0294] In summary, patients with lower overall disease burden or less prior chemotherapy may have a greater likelihood of a lasting response to brexu-cel, as those with sustained responses had lower tumor burdens, less prior platinum and cross-linking therapy, and higher CAR T cell proliferation. However, sustained responses were also observed in patients with high-risk disease characteristics.

[0295] Introduction Bruton's tyrosine kinase (BTK) inhibition has offered a paradigm shift in mantle cell lymphoma (MCL) therapy, but these drugs have not been proven curative. In patients with relapsed / refractory (R / R) MCL treated with BTK inhibitors, median progression-free survival (PFS) ranges from 13 to 33 months, and discontinuation of treatment due to progression or intolerance is common. The clinical benefit of BTK inhibitors is further limited in patients with high-risk features, including first progression within 24 months of initial diagnosis (POD24), TP53 abnormalities, elevated lactate dehydrogenase (LDH) at progression, and blastoid variants. Furthermore, outcomes of post-BTK inhibitor salvage therapy are poor, with median overall survival (OS) reported to be short, ranging from 2.5 to 8.4 months. Additionally, the BTKi ibrutinib was recently withdrawn from its MCL indication in the United States due to toxicity concerns. Therefore, there remains an unmet need for better treatment options for patients with R / R MCL.

[0296] Chimeric antigen receptor (CAR) T-cell therapy represents another landmark advance in the treatment of hematological malignancies. Brexcabutadiene autolucel (brexu-cel, formerly known as KTE-X19) is an autoanti-CD19 CAR T-cell therapy approved in the United States for the treatment of adults with R / R MCL and in the European Union for the treatment of adults with R / R MCL after two or more prior systemic therapies including BTK inhibitors. The rapid approval was based on the results of the central single-arm, multicenter phase 2 ZUMA-2 (NCT02601313) trial of brexu-cel therapy in patients with R / R MCL. All patients had progressed on BTK inhibitor therapy (62% were refractory) and many had high-risk conditions.

[0297] In the ZUMA-2 study, at a median follow-up of 35.6 months, the overall response rate (ORR) was 91% (95% CI, 81.8–96.7) among 68 treated patients, including a 68% complete response (CR) rate. The median duration of response (DOR) among responders was 28.2 months (95% CI, 13.5–47.1), which was considerably longer in patients achieving CR (46.7 months) than in patients with partial response (PR; 2.2 months). The median progression-free survival (PFS) and overall survival (OS) were 25.8 months (95% CI, 9.6–47.6) and 46.6 months (95% CI, 24.9–Not Estimated [NE]), respectively. At a median follow-up of 12.3 months, the most common grade 3 or higher adverse events (TEAEs) occurring under treatment were cytopenia with grade 3 or higher cytokine release syndrome (CRS) (94%) and infection (32%), respectively, and neurological events occurring in 15% and 31% of patients. Similar real-world results were observed from the US Lymphoma CAR T Consortium, which found that brexu-cel demonstrated a 90% overall response rate and an 82% complete response rate in 168 R / R MCL patients treated with brexu-cel in a standard treatment setting. Furthermore, this study found that 8% and 32% of these patients experienced grade 3 or higher CRS and neurotoxicity, respectively, after brexu-cel infusion.

[0298] Understanding the association between patient, product, and pharmacokinetic characteristics and persistent response to brexu-cel in patients with R / R MCL can inform prior patient selection and thereby maximize benefits. This study investigated the association between these factors and long-term response to brexu-cel in ZUMA-2.

[0299] method Research design and patients The detailed methodology for the multicenter, single-arm ZUMA-2 (NCT02601313) study has been previously described. Briefly, patients were 18 years of age or older and had histologically confirmed relapsed or refractory MCL to 1 to 5 previous MCL regimens, including chemotherapy with anthracyclines or bendamustine, anti-CD20 monoclonal antibodies, and BTK inhibitor therapy with either ibrutinib or acalabrutinib. All patients underwent leukocytosis, after which patients with high disease burden could receive bridging therapy with steroids or BTK inhibitors at the discretion of the principal investigator.

[0300] Pre-treatment chemotherapy consists of intravenous (IV) fludarabine 30 mg / m² once daily on days -5, -4, and -3. 2 and cyclophosphamide 500 mg / m² 2 It consisted of: a single IV infusion of brexu-cel, 2 x 10 on day 0. 6 The target dose of CAR T cells / kg was administered. All patients provided written informed consent, and the clinical trial was conducted in accordance with the principles of the Declaration of Helsinki.

[0301] Endpoints and evaluation This analysis examined baseline patient and disease characteristics, product characteristics, subsequent treatment, and pharmacological outcomes based on response status at 24 months after brexu-cel infusion, as assessed by an independent radiological review committee using the Lugano classification. Continued responders were defined as those with ongoing complete response (CR) or partial response (PR) at the 24-month evaluation. Relapsed responders were defined as those with a previous response who had relapsed, progressed to subsequent anticancer therapy (including sustained cancer therapy (SCT), or died for any cause before the 24-month evaluation. Non-responders were patients who did not achieve a response. Duration of response (DOR) was assessed in both responder groups. Levels of transduced anti-CD19 CAR T cells in the blood were measured by quantitative polymerase chain reaction. T cell phenotype was assessed by multicolor flow cytometry using previously described protocols and antibodies.

[0302] statistical analysis All subgroup analyses were post-hoc exploratory analyses, and descriptive statistics were provided. The Kaplan-Meier method was used to analyze endpoints between events.

[0303] result patient Of the 74 patients enrolled in ZUMA-2 and receiving leukocytapheresis, 71 (95.9%) successfully produced brexu-cel, and 68 (91.9%) received brexu-cel. As of July 24, 2021, the median follow-up was 35.6 months (range, 25.9–56.3), with 62 patients (91.2%) achieving the best response (CR) or PR, and 6 patients (8.8%) not achieving a response. Of the 62 responders, 29 (47%; 28 CRs and 1 PR) maintained their response at their 24-month evaluation (continued responders), 29 (47%; 15 CRs and 14 PRs) relapsed before the 24-month evaluation (relapsed responders), and 4 patients did not reach or missed the 24-month evaluation and were excluded from this analysis.

[0304] Patient characteristics Most baseline characteristics were similar across patients with continued responses, relapsed responses, and non-responses. However, patients with continued responses had approximately four times lower baseline tumor burden (median SPD 935 vs. 4861 mm) compared with patients with relapsed responses. 2 They were less likely to have received prior platinum therapy (10% vs. 41%) or bridging therapy (21% vs. 52%), less likely to have POD24 (33% vs. 66%), and more likely to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 at baseline (79% vs. 59%). Similar proportions of continuing and relapsed responders had received prior bendamustine (45% and 52%), prior anthracyclines (76% and 72%), and prior proteasome inhibitors (41% and 34%), respectively. These trends generally hold true when comparing only continuing responders with complete response (CR) to relapsed responders with complete response (CR).

[0305] The median number of prior therapies was 3 in all subgroups. Ibrutinib was the most common prior BTKi in all subgroups and the most common last prior therapy in all subgroups; however, it was more common among continuing responders (93% total; 66% as last prior therapy), relapsed responders (79% total; 41% as last prior therapy), or non-responders (67% total; 33% as last prior therapy). 28% of continuing responders, 21% of relapsed responders, and 33% of non-responders received acalabrutinib as a prior therapy; for 14% of continuing responders, 14% of relapsed responders, and 17% of non-responders, it was the last prior therapy. The median time from the last prior therapy to brexu-cel injection was 63 days (range, 26–748) for patients with continued responses, 63 days (range, 22–443) for patients with relapsed responses, and 136 days (range, 29–642) for patients without responses.

[0306] Effectiveness Among patients who achieved complete response (CR), the median duration of response (DOR) was not achieved in continuing response patients (n=28) (95% CI, 46.7–NE), and was 8.3 months (95% CI, 5.0–13.6) in relapsed response patients (n=15). At the data cutoff, of the 28 continuing response patients with CR, 22 (79%) remained in a state of continued response without further treatment, 1 (4%) progressed to a new anticancer therapy, 2 (7%) experienced disease progression, and 3 (11%) died. At the data cutoff, none of the 15 relapsed response patients with CR remained in a state of continued response without further treatment; 2 (13%) progressed to subsequent sustained response (SCT), 1 (7%) progressed to a new anticancer therapy, 12 (80%) experienced disease progression, and there were no deaths.

[0307] In patients who achieved any response (CR or PR), the median duration of response (DOR) was 47.1 months (95% CI, 36.5-NE) in continuing responders (n=29) and 5.0 months (95% CI, 2.2–8.3) in relapsed responders (n=29). The median time to response was 1 month for both continuing responders (range, 0.9–3.1; n=29) and relapsed responders (range, 0.8–1.7; n=29), and the median time to CR was 3 months for both continuing responders (range, 0.9–35.1; n=28) and relapsed responders (range, 0.8–9.0; n=15). As previously reported, MRD negativity at 6 months correlated with longer median DOR, PFS, and OS.

[0308] Considering that elevated LDH is a known indicator of poor prognosis in R / R MCL patients, DOR was evaluated in patients with high and low baseline LDH levels. The median DOR for patients with high baseline LDH (≥1 ULN) (n=13) and low baseline LDH (<1 ULN) (n=14) was 47.1 months (95% CI, 24.8-NE) and 46.7 months (95% CI, 24.4-NE), respectively. For relapsed responders with high baseline LDH (n=11) and low baseline LDH (n=18), the median DOR was 3.6 months (95% CI, 1.0–13.5) and 5.4 months (95% CI, 2.2–8.6), respectively.

[0309] Subsequent chemotherapy was administered to 1 / 29 (3%), 20 / 29 (69%), and 3 / 6 (50%) of patients in the subgroups of those who responded to continued cancer, those who responded to relapsed cancer, and those who did not respond to cancer. The most common of these were radiotherapy, rituximab, dexamethasone, lenalidomide, and venetoclax.

[0310] Product and pharmacokinetic characteristics Product characteristics were generally similar between patients with continued responses and those with relapsed responses. Numerical differences were observed between the median CD4 / CD8 ratios of 0.86 (range, 0.27–2.06), 0.64 (range, 0.04–3.73), and 0.41 (range, 0.25–0.73), respectively, for patients with continued responses, those with relapsed responses, and those without responses. While the total number of injected CAR T cells was similar across subgroups, the total number of injected CCR 7+ T cells was moderately increased among patients with continued responses compared to other subgroups, with a median level (×10⁻⁶). 6 The values ​​were 119.8 (range 37.0-249.9), 89.4 (range 6.1-353.4), and 88.2 (range 39.9-150.3), respectively.

[0311] The median peak CAR T cell levels were 102.4 cells / μl (range 0.3–2242.6) and 62.7 cells / μl (range 1.6–2589.5) in patients with continued responses and those with relapsed responses, respectively. Similarly, the median area under the curve (AUC) of CAR T cells (days 0–28) was also measured. 0-28 The values ​​for these subgroups were 1487.0 cells / μl × day (range, 3.8–16700) and 775.8 cells / μl × day (range, 19.0–27200), respectively. Non-responders had the lowest median CAR T cell peak (5.9 cells / μl; range, 0.2–95.9) and AUC for any subgroup. 0-28 The values ​​were (24.7 cells / μl × day; range, 1.8~1089.1).

[0312] Differences in peripheral blood T cell phenotype on day 7 were observed between subgroups, including those with significantly higher median percentages of differentiated CD8+CD27-CD28+ cells and CD8+CCR7-CD45RA+CD27-CD28+ terminally differentiated effector memory cells in relapsed and non-response patients combined, compared to continuing responders (P=0.0023 and P=0.0052, respectively; Table 12). Continuing responders had significantly more (P=0.03) peripheral CD4+ T cells maintaining juvenile CD27+ expression than relapsed and non-response patients combined, and showed a tendency toward higher levels of activated CD8 effector memory T cells (P=0.057; Table 12).

[0313] TIFF2026508791000030.tif251170TIFF2026508791000031.tif54170

[0314] Consideration This analysis identifies the association between patient, disease, product, and / or pharmacokinetic characteristics and persistent response to Brexu-cel in patients with R / R MCL treated with ZUMA-2. The analysis identified a 47% rate of continued response at 24 months post-infusion. Given the poor prognosis and limited survival associated with BTK inhibitor failure in this situation, these results continue to support brexu-cel as a preferred treatment option in this disease state. Interestingly, the median DOR for relapsed responders was only 5 months, while the median DOR for continued responders was 47.1 months, suggesting that patients who maintained a response beyond 24 months had a favorable long-term outcome. Notably, the significant difference in the Kaplan-Meier DOR curves between continued responders and relapsed responders between 6 and 12 months suggests that these earlier time points may predict long-term responses.

[0315] Because CAR T-cell therapy is a relatively recent approach to treating patients with hematological malignancies, factors associated with response are subject to ongoing investigation. These factors may be specific to a given CAR-T cell product and / or malignancy, and definitive data are still lacking. Patient characteristics such as baseline tumor burden and LDH levels, as well as T-cell phenotypes such as the percentage of memory T cells and the CD4+ / CD8+ T-cell ratio, have correlated with response or response persistence to CD-19 targeted CAR T-cell therapy. More recently, tumor immune status has been suggested to be a determinant of CAR-T cell efficacy and may also play a role in response persistence in this analysis.

[0316] In ZUMA-2, the tumor burden, as measured by SPD at baseline, was significantly lower in patients with continued responses than in patients with relapsed responses (935 vs. 4861 mm²). 2 A similar correlation between SPD and outcome has been reported in another study of CAR T-cell therapy in B-cell malignancies. Prior platinum therapy and bridging therapy were less frequent in continuing responders compared to relapsed responders, and continuing responders were more likely to have a better ECOG performance status than relapsed responders. Taken together, these data suggest that, among patient and disease characteristics, a higher tumor burden at baseline was associated with a higher risk of relapse up to 24 months. In contrast, patients with low and high baseline LDH within each subgroup (continuing responders or relapsed responders) had similar median DORs, indicating that a known risk factor of high baseline LDH levels (above the upper limit of normal) was not associated with inferior response persistence. Therefore, brexu-cel was associated with persistent response regardless of baseline LDH status.

[0317] Ibrutinib was administered more frequently as a pre-treatment and last-line pre-treatment in patients with ongoing responses than in patients with relapsed or non-responses. Interestingly, the same phenomenon was not observed with prior acalabrutinib therapy, as similar proportions of patients with ongoing responses, relapsed responses, and non-responses received acalabrutinib as a pre-treatment or last-line pre-treatment. In preclinical studies, ibrutinib has been shown to improve the persistence and efficacy of CAR-T cells, possibly by improving T cell function and proliferation through its off-target inhibition of inducible T cell kinases. In addition, a time-limited combination of ibrutinib and tisagenlecleucel showed promising efficacy (ORR 90%) in a small patient population (N=20) with R / R MCL in the TARMAC trial. Further clinical studies are needed to better understand the potential impact of prior ibrutinib on the long-term efficacy of brexu-cel therapy.

[0318] Previous analyses of ZUMA-2 showed that prior bendamustine use within 6 months of apheresis was associated with a reduced pharmacokinetic profile of injected CAR T cells and a shortened product doubling time, suggesting that the timing of bendamustine use may weaken T cell compatibility; however, the small sample size limits the interpretation of these findings. In this analysis, the rate of prior bendamustine use was similar between patients with continuous responses and those with relapsed responses; however, due to the small sample size, it was not possible to assess whether the timing of preceding bendamustine use affects the durability of the response.

[0319] CAR T products derived from patients with different T cell subsets can reasonably be expected to have different effects. In ZUMA-2, T cell phenotypic data were available for most patients, allowing for comparison of T cell subsets between continuing responders and relapsed / non-responders combined. The increased median number of CCR7+ cells and CD27+ peripheral T cells in continuing responders suggests that sustained memory T cell differentiation may play a role in achieving lasting response.

[0320] Previous studies of axicabtagene ciloleucel (ZUMA-1) found that peak CAR T cell levels and CAR T cell AUC correlated with persistent (24-month) response. This analysis identified a similar trend in R / R MCL patients treated with brexu-cel in ZUMA-2. Both peak median CAR T cell levels and AUC levels were approximately twice as high in patients with sustained responses, suggesting that strong CAR T cell proliferation may contribute to achieving persistent response. These findings are consistent with results from ZUMA-7 (e.g., JULIET), even though this effect was not observed in other CAR T studies using different CAR T products. Factors specific to disease context, tumor characteristics including the microenvironment, patient baseline characteristics, and product characteristics may prevent extrapolation or generalization.

[0321] These results demonstrate a persistent response in patients with R / R MCL treated with brexu-cel (including, if not treated, patients with high-risk disease features resulting in a poor prognosis). Several factors that appear to be associated with sustained response for two years (i.e., ECOG performance status, SPD, and prior platinum and bridging therapy use) were related to disease progression or aggressiveness, suggesting that a more optimal use of brexu-cel may be possible at an earlier stage of disease progression. Consistent with some, but not all, prior CAR T studies, the degree of CAR T cell proliferation was associated with persistence of response. These findings, along with findings from further investigation, should help identify patients who are likely to benefit most from brexu-cel in this difficult-to-treat malignancy.

[0322] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent as each individual publication, patent, patent application, or other document is individually indicated to be incorporated herein by reference for all purposes.

[0323] While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure.

Claims

1. A method for treating mantle cell lymphoma (MCL) or B-cell ALL in a subject requiring treatment of the mantle cell lymphoma or B-cell ALL, comprising administering to the subject a T-cell product containing autologous T cells expressing a therapeutically effective amount of anti-CD19 chimeric antigen receptor (CAR), wherein the MCL or B-cell ALL is relapsed or refractory MCL after one or more prior treatments selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation (SCT), or any combination thereof, and further comprising one or more prior treatments that do not include Bruton's tyrosine kinase inhibitors (BTKi).

2. The method according to claim 1, wherein the subject has received one to five prior treatments, and at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibody, and / or chemotherapy comprising anthracycline or bendamustine.

3. The method according to claim 1 or 2, wherein the BTKi is ibrutinib or acalabrutinib.

4. The method according to any one of claims 1 to 3, wherein R / R B-cell ALL is defined as resistance to first-line therapy (i.e., resistance to primary treatment), relapse within 12 months of initial remission, relapse or refractory after two or more prior systemic therapies, or relapse after allogeneic SCT, and the subject is required to have a myeloblast percentage of 5% or more, an Eastern Cooperative Oncology Group performance status of 0 or 1, and / or sufficient renal, hepatic, and cardiac function.

5. The method according to any one of claims 1 to 4, wherein, if the subject of the B-cell ALL has been administered prior to blinatumomab, the subject is required to have leukemic blasts with CD19 expression of ≥90%.

6. The method according to any one of claims 1 to 5, wherein the subject receives bridging therapy after leukocyte apheresis and before pretreatment / lymphocyte apheresis chemotherapy.

7. The aforementioned MCL target is intravenous cyclophosphamide 500 mg / m². ２ and intravenous fludarabine 30 mg / m² ２ The method according to any one of claims 1 to 6, wherein the patient receives a lymphocyte-depleting chemotherapy regimen, both of which are administered 5 days, 4 days, and 3 days prior to T-cell infusion, respectively.

8. The B-cell ALL subjects were administered intravenously (IV) fludarabine 25 mg / m² four, three, and two days prior to T-cell injection. ２ / day, and IV cyclophosphamide 900 mg / m³ two days before the infusion. ２ The method according to any one of claims 1 to 7, wherein the lymphocyte removal regimen is performed on a daily basis.

9. The method according to claim 6 or 8, wherein the MCL bridging therapy is selected from dexamethasone (e.g., PO or IV 20-40 mg equivalent, once daily for 1-4 days); methylprednisolone, ibrutinib (e.g., PO 560 mg, once daily), and / or acalabrutinib (e.g., PO 100 mg, twice daily); immunomodulators; R-CHOP, bendamustine; alkylating agents; and / or platinum-based agents, and the bridging therapy is administered after leukocyte apheresis and completed, for example, within 5 days prior to pre-treatment chemotherapy.

10. The method according to any one of claims 6 to 8, wherein the subject of the B cell ALL can receive one or more of the following bridging chemotherapy regimens. Table 1

11. The method according to any one of claims 1 to 10, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

12. The method according to claim 11, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3 zeta domains.

13. The method according to claim 11 or 12, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from a leukocyte apheresis product in which positive selection for CD4+ and CD8+ T cells has not been performed.

14. The method according to any one of claims 11 to 13, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from a leukocyte apheresis-derived product that has not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

15. The method according to claim 14, wherein the superior product characteristics are selected from an increase in the proportion of CDRA45+CCR7+ (naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and / or a decrease in the proportion of CD3- cells.

16. The target of the MCL is 1.8×10 ６ , 1.9×10 ６ , or 2×10 ６ viable CAR-positive T cells per kg of body weight, and up to 2×10 ８ viable CAR-positive T cells (in the case of patients weighing 100 kg or more) are administered one or more times. The target of the B cell ALL is 0.5×10 ６ , 1×10 ６ , or 2×10 ６ viable CAR-positive T cells, and up to 2×10 ８ viable CAR-positive T cells (in the case of patients weighing 100 kg or more) are administered. The method according to any one of claims 1 to 15.

17. The method according to any one of claims 1 to 15, wherein if the subject achieves a complete response to the initial injection, the subject may receive a second injection of anti-CD19 CAR T cells, provided that if the condition worsens after more than three months of remission, CD19 expression is maintained and there is no suspicion of neutralizing antibodies against CAR, and the response is evaluated using the Lugano classification.

18. The method according to any one of claims 1 to 17, wherein after T cell administration, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity.

19. The method according to claim 18, wherein the subject is monitored daily for at least 7 days, preferably 4 weeks, after injection for signs and symptoms of CRS and neurotoxicity.

20. The method according to claim 18 or 19, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and / or hypotension, and the signs or symptoms associated with neurological events include encephalopathy, seizures, altered level of consciousness, speech disorder, tremor, and / or confusion.

21. The method according to any one of claims 18 to 20, wherein cytokine release syndrome in a patient with MCL is managed according to the following protocol. Table 2

22. The method according to any one of claims 18 to 21, wherein neurotoxicity in subjects of MCL is controlled according to the following protocol. Table 3

23. The method according to any one of claims 1 to 22, wherein the subject of the MCL is a high-risk patient determined by a Ki-67 tumor growth index of 50% or more and / or the presence of a TP53 mutation.

24. The method according to any one of claims 18 to 20, wherein CRS in a subject of B cells ALL is controlled according to the following protocol. Table 4

25. The method according to any one of claims 18 to 20 and 24, wherein neurotoxicity in a B-cell ALL subject is controlled according to the following protocol. Table 5

26. The method according to any one of claims 1 to 25, wherein the subject of the B cell ALL can receive one or more of the following bridging chemotherapy regimens. Table 6

27. Autologous T cells expressing anti-CD19 CAR for use in a method for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of claims 1 to 26.

28. The use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of claims 1 to 26.

29. A method for treating mantle cell lymphoma (MCL) in a subject requiring treatment for MCL, comprising administering to the subject a therapeutically effective amount of a T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), wherein the MCL is relapsed or refractory MCL and the last prior therapy occurred less than 60 months prior to the administration of the T cell product.

30. The method according to claim 29, wherein the MCL is refractory to or has relapsed after one or more of the following: chemotherapy, radiotherapy, immunotherapy (including T-cell therapy and / or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof.

31. The method according to claim 29 or 30, wherein the subject has received one to three prior treatments, and at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibody, anthracycline or bendamustine chemotherapy, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

32. The method according to claim 31, wherein the BTKi is ibrutinib.

33. The method according to claim 32, wherein ibrutinib was the last treatment administered before the administration of the T-cell product.

34. The method according to any one of claims 29 to 33, wherein the subject has undergone leukocyte apheresis and has not received bridging therapy prior to pretreatment / lymphocyte apheresis chemotherapy.

35. The method according to any one of claims 29 to 34, wherein the subject has not undergone prior platinum therapy.

36. The aforementioned subjects received intravenous cyclophosphamide 500 mg / m². ２ and intravenous fludarabine 30 mg / m² ２ The method according to any one of claims 29 to 35, wherein the patient receives a lymphocyte-depleting chemotherapy regimen, both of which are administered five days, four days, and three days prior to T-cell infusion, respectively.

37. The method according to any one of claims 29 to 36, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

38. The method according to claim 37, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3 zeta domains.

39. The method according to claim 37 or 38, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from leukocyte apheresis-derived products in which positive selection for CD4+ and CD8+ T cells has not been performed.

40. The method according to any one of claims 37 to 39, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from a leukocyte apheresis-derived product that has not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

41. The method according to claim 40, wherein the aforementioned excellent product characteristics are selected from an increase in the proportion of CDRA45+CCR7+ (naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and a decrease in the proportion of CD3- cells.

42. For the aforementioned subjects, 1.8 × 10 per kg of body weight ６ piece, 1.9×10 ６ individual or 2 x 10 ６ One or more doses of CAR-positive live T cells are administered, up to a maximum of 2 × 10⁶ ８ The method according to any one of claims 29 to 41, wherein 100 CAR-positive live T cells (in the case of a patient weighing 100 kg or more) are administered.

43. The method according to any one of claims 29 to 42, wherein if the subject achieves a complete response to the initial injection, the subject may receive a second injection of anti-CD19 CAR T cells, provided that if the condition worsens after more than three months of remission, CD19 expression is maintained and there is no suspicion of neutralizing antibodies against CAR, and the response is evaluated using the Lugano classification.

44. The method according to any one of claims 29 to 43, wherein, after T-cell administration, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity.

45. The method according to claim 44, wherein the subject is monitored daily for at least 7 days, preferably 4 weeks, after injection for signs and symptoms of CRS and neurotoxicity.

46. The method according to claim 44 or 45, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and / or hypotension, and the signs or symptoms associated with neurotoxicity include encephalopathy, seizures, altered level of consciousness, speech disorder, tremor, and / or confusion.

47. The method according to any one of claims 44 to 46, wherein cytokine release syndrome in a subject of MCL is managed according to the following protocol. Table 7

48. The method according to any one of claims 44 to 47, wherein neurotoxicity in the subject of MCL is controlled according to the following protocol. Table 8

49. The method according to any one of claims 29 to 48, wherein the subject is a high-risk patient determined by a Ki-67 tumor growth index of 50% or more and / or the presence of a TP53 mutation.

50. Autologous T cells expressing anti-CD19 CAR for use in a method for treating MCL according to any one of claims 29 to 49.

51. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating MCL according to any one of claims 29 to 50.

52. A method for treating a cancer selected from the group consisting of Waldenström macroglobulinemia, Richter transformation, Burkitt lymphoma, and pilaris cell leukemia in a subject requiring treatment for such cancer, comprising administering to the subject a T-cell product containing autologous T cells expressing a therapeutically effective amount of anti-CD19 chimeric antigen receptor (CAR), wherein the subject receives bridging therapy after leukocyte apheresis and before pre-treatment / lymphocyte apheresis chemotherapy.

53. The method according to claim 52, wherein the cancer is refractory to one or more of the following: chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation, or any combination thereof, or has subsequently recurred.

54. The method according to claim 52 or 53, wherein the bridging therapy is completed at least 7 days or 5 half-lives before the pre-treatment chemotherapy.

55. The aforementioned subjects received intravenous cyclophosphamide 500 mg / m². ２ and intravenous fludarabine 30 mg / m² ２ The method according to any one of claims 52 to 54, wherein the patient receives a lymphocyte apheresis chemotherapy regimen, both of which are administered five days, four days, and three days prior to T cell infusion, respectively.

56. The cancer is Richter transformation, and the bridging therapy is rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine and prednisone (R-CHOP); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R); Bruton's tyrosine kinase inhibitor (BTKi) (BTKi) ± VTX-2337; dexamethasone; and irradiation. A method according to any one of claims 52 to 55, selected from the group consisting of the following.

57. The cancer is Burkitt lymphoma, and the bridging therapy is rituximab, ifosfamide, carboplatin and etoposide (R-ICE); dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R); rituximab, gemcitabine and oxaliplatin (R-GEMOX); cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride and dexamethasone (HyperCVAD); dexamethasone; and irradiation. A method according to any one of claims 52 to 55, selected from the group consisting of the following.

58. The method according to any one of claims 52 to 55, wherein the cancer is Waldenström macroglobulinemia and the bridging therapy is ibrutinib.

59. The method according to any one of claims 52 to 58, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and the resulting partial or complete depletion of circulating cancer cells.

60. The method according to claim 59, wherein the PBMC is enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a non-replicating viral vector containing FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3 zeta domains.

61. The method according to claim 59 or 60, wherein the T cell product contains fewer cancer cells than a T cell product containing T cells from leukocyte apheresis-derived products in which positive selection for CD4+ and CD8+ T cells has not been performed.

62. The method according to any one of claims 59 to 61, wherein the T cell product has other superior product characteristics compared to a T cell product containing T cells from a leukocyte apheresis-derived product that has not undergone positive selection / enrichment for CD4+ and CD8+ T cells.

63. The method according to claim 62, wherein the aforementioned excellent product characteristics are selected from an increase in the proportion of CDRA45+CCR7+ (naive-like) T cells, a decrease in the proportion of differentiated T cells, an increase in the proportion of CD3+ cells, a decrease in IFN-gamma production, and a decrease in the proportion of CD3- cells.

64. For the aforementioned subjects, 1.8 × 10 per kg of body weight ６ piece, 1.9×10 ６ individual or 2 x 10 ６ One or more doses of CAR-positive live T cells are administered, up to a maximum of 2 × 10⁶ ８ The method according to any one of claims 52 to 63, wherein 100 CAR-positive live T cells (in the case of a patient weighing 100 kg or more) are administered.

65. The method according to any one of claims 52 to 64, wherein after T cell administration, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity.

66. The method according to claim 65, wherein the subject is monitored daily for at least 7 days, preferably 4 weeks, after injection for signs and symptoms of CRS and neurotoxicity.

67. The method according to claim 65 or 66, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and the signs or symptoms associated with neurological events include encephalopathy, seizures, altered level of consciousness, speech disorders, tremors, and confusion.

68. Autologous T cells expressing anti-CD19 CAR for use in a method for treating cancer according to any one of claims 52 to 67.

69. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a pharmaceutical product for treating cancer according to any one of claims 52 to 67.

70. A method for treating cancer in a subject requiring cancer treatment, wherein the subject has previously been administered a first T-cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), and further, a peripheral blood sample is collected from the subject after administration of the first T-cell product. (a) Measuring the level of CD8+CD27-CD28+ T cells in the blood sample, (b) A method comprising administering a second T cell product to a subject if the level of CD8+CD27-CD28+ T cells in a blood sample is elevated.

71. A method for treating cancer in a subject requiring cancer treatment, wherein the subject has previously been administered a first T-cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), and further, a peripheral blood sample is collected from the subject after administration of the first T-cell product. (a) Measuring the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, (b) A method comprising administering a second T cell product to a subject if the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in a blood sample is elevated.

72. The method according to claim 70 or 71, wherein the first T cell product comprises CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and resulting partial or complete depletion of circulating cancer cells.

73. The method according to claim 72, wherein the CD4+ and CD8+ T cells are activated with an anti-CD3 antibody and an anti-CD28 antibody in the presence of IL-2, and then transduced with a non-replicating viral vector encoding a chimeric antigen receptor (CAR) comprising an anti-CD19 single-strand variable fragment (scFv), CD28, and CD3 zeta domains.

74. The method according to any one of claims 70 to 73, wherein the cancer is selected from the group consisting of mantle cell lymphoma (MCL), B-cell ALL, Waldenström macroglobulinemia, Richter transformation, Burkitt lymphoma, and pilaris cell leukemia.

75. The method according to claim 74, wherein the cancer is MCL.

76. The method according to any one of claims 70 to 75, wherein the blood sample is collected from the subject between the fifth and ninth day after administration of the first T cell product.

77. The method according to claim 76, wherein the blood sample is collected from the subject between the 6th and 8th day after administration of the first T cell product.

78. The method according to claim 77, wherein the blood sample is collected from the subject seven days after administration of the first T cell product.

79. The method according to any one of claims 70 to 75, wherein the blood sample is collected from the subject between 12 and 16 days after administration of the first T cell product.

80. The method according to claim 79, wherein the blood sample is collected from the subject between 13 and 15 days after administration of the first T cell product.

81. The method according to claim 80, wherein the blood sample is collected from the subject 14 days after administration of the first T cell product.

82. The method according to claim 70, wherein the elevated level of CD8+CD27-CD28+ T cells in the subject is determined by comparison with other subjects who have been administered an equivalent T cell product and whose peripheral blood samples were collected on the same day after administration of the T cell product.

83. The method according to claim 71, wherein the elevated level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the subject is determined by comparison with other subjects who have been administered an equivalent T cell product and who have had peripheral blood samples taken on the same day after administration of the T cell product.

84. The method according to any one of claims 70 to 83, wherein the second T cell product is selected from the group consisting of autologous CD19 / CD20 bisistronic T cell products and allogeneic T cell products.

85. A T-cell product for use in a method for treating cancer according to any one of claims 70 to 84.

86. Use of a T-cell product in the manufacture of a pharmaceutical product for treating cancer according to any one of claims 70 to 84.

87. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administering the first T cell product, (b) Measuring the level of CD8+CD27-CD28+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of CD8+CD27-CD28+ T cells in the blood sample, wherein if the level of CD8+CD27-CD28+ T cells is elevated, a second T cell product is administered.

88. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administering the first T cell product, (b) Measuring the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, wherein if the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells is elevated, a second T cell product is administered.

89. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administering the first T cell product, (b) Measuring the level of CD27+CD28-CD4+CD3+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of CD27+CD28-CD4+CD3+ T cells in the blood sample, wherein if the level of CD27+CD28-CD4+CD3+ T cells is elevated, the second T cell product is not administered.

90. A method for monitoring subjects who have previously received a first T cell product containing autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR), (a) Collecting a blood sample from the subject after administering the first T cell product, (b) Measuring the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells in the blood sample, (c) A method comprising prescribing a series of treatments based on the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells in the blood sample, wherein if the level of PD1+CCR7+CD45RA-CD8+CD3+ T cells is elevated, the series of treatments is prescribed without administering a second T cell product.

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