Improving efficacy and persistent response of immunotherapy

Abstract

Provided herein are methods for preparing, producing, processing, culturing, isolating or manufacturing cells suitable for immune or cell therapy and their use in cell therapy. Further provided are methods of treating cancer patients with such cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 381,525, filed on October 28, 2022, and entitled “Efficacy and Durable Response of Immunotherapy,” U.S. Provisional Patent Application No. 63 / 386,457, filed on December 7, 2022, and entitled “Efficacy and Durable Response of Immunotherapy,” U.S. Provisional Patent Application No. 63 / 479,877, filed on January 13, 2023, and entitled “Efficacy and Durable Response of Immunotherapy,” and U.S. Provisional Patent Application No. 63 / 515,492, filed on July 25, 2023, and entitled “Efficacy and Durable Response of Immunotherapy,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to CAR-T cells, methods for preparing the same, and methods for using the same to treat cancer. Background Art

[0004] Human cancer is essentially composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. Cancer cells express proteins and other antigens that are different from those expressed by normal cells. The body's innate immune system can use these abnormal tumor antigens to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells (such as T and B lymphocytes) from successfully targeting cancer cells. Human T cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in subjects (e.g., patients). Various techniques have been developed to prepare T cell populations with enriched concentrations of naturally occurring T cells capable of targeting tumor antigens; remove circulating tumor cells; and / or genetically modify T cells to specifically target known cancer antigens, thereby producing a population of chimeric antigen receptor (CAR)-T cells for cancer therapy. Some of these therapies have shown promising effects on tumor size and patient survival. There is a need for treatment methods that utilize (CAR)-T cells for cancer therapy, including methods that include bridging therapy. Summary of the invention

[0005] Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. Although the present invention has been described in conjunction with specific embodiments of the present invention, this description is intended to illustrate rather than limit the scope of the present invention, which is defined in part by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following embodiments / claims.

[0006] 1. A method for treating mantle cell lymphoma (MCL) or B-cell ALL in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), wherein the MCL or B-cell ALL is relapsed or refractory MCL after one or more previous treatments selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation (SCT), or any combination thereof, further wherein the one or more previous treatments do not comprise a Bruton's tyrosine kinase inhibitor (BTKi).

[0007] 2. The method according to aspect 1, wherein the subject has received 1 to 5 previous treatments, wherein at least one of the previous treatments is selected from autologous SCT, anti-CD20 antibodies and / or chemotherapy containing anthracyclines or bendamustine.

[0008] 3. The method according to aspect 1 or 2, wherein the BTKi is ibrutinib or acalabrutinib.

[0009] 4. The method of any one of aspects 1 to 3, wherein R / RB cell ALL is defined as refractory to first-line therapy (i.e., primary refractory), relapses ≤12 months after first remission, relapses or is refractory after ≥2 prior lines of systemic therapy, or relapses after allogeneic SCT, wherein the subject is required to have ≥5% bone marrow blasts, an Eastern Cooperative Oncology Group performance status of 0 or 1, and / or adequate renal, hepatic, and cardiac function.

[0010] 5. The method according to any one of aspects 1 to 4, wherein if the B-cell ALL subject has received prior blinatumomab, the subject is required to have leukemic blasts with CD19 expression ≥90%.

[0011] 6. The method according to any one of aspects 1 to 5, wherein the subject receives bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

[0012] 7. The method according to any one of aspects 1 to 6, wherein the MCL subject receives 500 mg / m 2 intravenously administered on each of the fifth, fourth, and third days prior to T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously2 Fludarabine and both lymphodepleting chemotherapy regimens.

[0013] 8. The method according to any one of aspects 1 to 7, wherein the B-cell ALL subject receives 25 mg / m 2 per day administered intravenously (IV) on each of the fourth, third, and second days prior to T cell infusion. 2 Fludarabine plus 900 mg / m2 / day given IV on the day before infusion 2 Lymphodepletion regimen with cyclophosphamide.

[0014] 9. The method of any one of aspects 6 or 8, wherein the MCL bridging therapy is selected from dexamethasone (e.g., 20 mg to 40 mg or equivalent given PO or IV daily for 1 to 4 days); methylprednisolone, ibrutinib (e.g., 560 mg given PO daily) and / or acalabrutinib (e.g., 100 mg given PO twice daily); an immunomodulator; R-CHOP, bendamustine; an alkylating agent; and / or a platinum-based agent, wherein the bridging therapy is administered after leukapheresis and is completed 5 days or less prior to, for example, conditioning chemotherapy.

[0015] 10. The method according to any one of aspects 6 to 8, wherein the B-cell ALL subject may receive any one or more of the following bridging chemotherapy regimens:

[0016]

[0017] 11. A method according to any one of aspects 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 subsequent partial or complete depletion of circulating cancer cells.

[0018] 12. A method according to aspect 11, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 antibodies and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing FMC63-28Z CAR (a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-ζ domains).

[0019] 13. The method of aspect 11 or 12, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

[0020] 14. A method according to any one of aspects 11 to 13, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product, wherein the leukapheresis-derived product has not been positively selected / enriched for CD4+ and CD8+ T cells.

[0021] 15. The method according to aspect 14, wherein the superior product attribute is selected from increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production and / or decreased percentage of CD3- cells.

[0022] 16. The method according to any one of aspects 1 to 15, wherein 1.8×10 6 pcs, 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients 100 kg and above), and 0.5×10 6 pcs, 1×10 6 or 2×10 6 CAR-positive live T cells / kg body weight, with the maximum value of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

[0023] 17. A method according to any one of aspects 1 to 15, wherein if the subject has achieved a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells if progression occurs after remission for >3 months, provided that CD19 expression has been retained and neutralizing antibodies against the CAR are not suspected, wherein the response is assessed using the Lugano classification.

[0024] 18. The method according to any one of aspects 1 to 17, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity following T cell administration.

[0025] 19. The method of aspect 18, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

[0026] 20. The method of aspect 18 or 19, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and / or hypotension, and signs or symptoms associated with neurological events include encephalopathy, seizures, changes in level of consciousness, speech disorders, tremors and / or confusion.

[0027] 21. The method according to any one of aspects 18 to 20, wherein cytokine release syndrome in a subject with MCL is managed according to the following regimen:

[0028]

[0029] 22. The method according to any one of aspects 18 to 21, wherein neurotoxicity in the MCL subject is managed according to the following regimen:

[0030]

[0031] 23. The method according to any one of aspects 1 to 22, wherein the MCL subject is a high-risk patient determined by the presence of a Ki-67 tumor proliferation index ≥ 50% and / or a TP53 mutation.

[0032] 24. The method according to any one of aspects 18 to 20, wherein CRS in a subject with B-cell ALL is managed according to the following regimen:

[0033]

[0034] 25. The method according to any one of aspects 18 to 20 and 24, wherein neurotoxicity in a subject with B-cell ALL is managed according to the following regimen:

[0035]

[0036]

[0037] 26. The method according to any one of aspects 1 to 25, wherein the B-cell ALL subject may receive any one or more of the following bridging chemotherapy regimens:

[0038]

[0039] 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 aspects 1 to 26.

[0040] 28. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a medicament for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of aspects 1 to 26.

[0041] 29. A method for treating mantle cell lymphoma (MCL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), wherein the MCL is relapsed or refractory MCL and the last prior therapy was less than 60 months prior to administration of the T cell product.

[0042] 30. The method of aspect 29, wherein the MCL is refractory to, or has relapsed following, one or more of chemotherapy, radiotherapy, immunotherapy (including T cell therapy and / or treatment with an antibody or antibody-drug conjugate), autologous stem cell transplantation, or any combination thereof.

[0043] 31. The method according to any one of aspects 29 or 30, wherein the subject has received 1 to 3 prior therapies, wherein at least one of the prior therapies is selected from autologous SCT, anti-CD20 antibodies, anthracycline- or bendamustine-containing chemotherapy, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

[0044] 32. The method according to aspect 31, wherein the BTKi is ibrutinib.

[0045] 33. The method of aspect 32, wherein ibrutinib is the last treatment prior to administration of the T cell product.

[0046] 34. The method according to any one of aspects 29 to 33, wherein the subject has not received bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

[0047] 35. The method according to any one of aspects 29 to 34, wherein the subject has not received prior platinum therapy.

[0048] 36. The method of any one of aspects 29 to 35, wherein the subject receives 500 mg / m 2 administered intravenously on each of the fifth, fourth, and third days prior to the T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously 2 Fludarabine and both lymphodepleting chemotherapy regimens.

[0049] 37. The method of any one of aspects 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 subsequent partial or complete depletion of circulating cancer cells.

[0050] 38. A method according to aspect 37, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 antibodies and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing FMC63-28Z CAR (a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-ζ domains).

[0051] 39. The method of aspect 37 or 38, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

[0052] 40. The method of any one of aspects 37 to 39, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

[0053] 41. The method according to aspect 40, wherein the superior product attribute is selected from the group consisting of increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production, decreased percentage of CD3- cells.

[0054] 42. The method according to any one of aspects 29 to 41, wherein 1.8×10 6 , 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

[0055] 43. A method according to any one of aspects 29 to 42, wherein if the subject has achieved a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells if progression occurs after remission for >3 months, provided CD19 expression has been retained and neutralizing antibodies against the CAR are not suspected, wherein the response is assessed using the Lugano classification.

[0056] 44. The method of any one of aspects 29 to 43, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity following T cell administration.

[0057] 45. The method of aspect 44, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

[0058] 46. ​​The method of any one of aspects 44 and 45, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and / or hypotension, and signs or symptoms associated with neurotoxicity include encephalopathy, seizures, changes in level of consciousness, speech disorders, tremors and / or confusion.

[0059] 47. The method according to any one of aspects 44 to 46, wherein cytokine release syndrome in the MCL subject is managed according to the following regimen:

[0060]

[0061] 48. The method according to any one of aspects 44 to 47, wherein neurotoxicity in the MCL subject is managed according to the following regimen:

[0062]

[0063] 49. The method according to any one of aspects 29 to 48, wherein the subject is a high-risk patient determined by the presence of a Ki-67 tumor proliferation index ≥ 50% and / or a TP53 mutation.

[0064] 50. Autologous T cells expressing anti-CD19 CAR for use in a method for treating MCL according to any one of aspects 29 to 49.

[0065] 51. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a medicament for treating MCL according to any one of aspects 29 to 50.

[0066] 52. A method for treating a cancer selected from the group consisting of Waldenstrom Macroglobulinemia, Richter Transformation, Burkitt Lymphoma, and Hairy Cell Leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), wherein the subject receives bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

[0067] 53. The method of aspect 52, wherein the cancer is refractory to, or has relapsed following, one or more of chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation, or any combination thereof.

[0068] 54. The method of aspect 52 or 53, wherein the bridging therapy is completed for ≥7 days or ≥5 half-lives prior to the conditioning chemotherapy.

[0069] 55. The method of any one of aspects 52 to 54, wherein the subject receives 500 mg / m 2 administered intravenously on each of the fifth, fourth, and third days prior to the T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously 2 Fludarabine and both lymphodepleting chemotherapy regimens.

[0070] 56. The method of any one of aspects 52 to 55, wherein the cancer is Reye's 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 radiation.

[0071] 57. The method of any one of aspects 52 to 55, wherein the cancer is Burkitt's 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 radiation.

[0072] 58. The method of any one of aspects 52 to 55, wherein the cancer is Waldenstrom's macroglobulinemia and the bridging therapy is ibrutinib.

[0073] 59. The method of any one of aspects 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 subsequent partial or complete depletion of circulating cancer cells.

[0074] 60. A method according to aspect 59, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-2, and then transduced with a replication-defective viral vector containing FMC63-28Z CAR (a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-ζ domains).

[0075] 61. The method of aspect 59 or 60, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

[0076] 62. The method of any one of aspects 59 to 61, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

[0077] 63. The method according to aspect 62, wherein the superior product attribute is selected from increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production, decreased percentage of CD3- cells.

[0078] 64. The method according to any one of aspects 52 to 63, wherein 1.8×10 6 , 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

[0079] 65. The method of any one of aspects 52 to 64, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity following T cell administration.

[0080] 66. The method of aspect 65, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

[0081] 67. The method of aspect 65 or 66, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and hypotension, and signs or symptoms associated with neurological events include encephalopathy, seizures, changes in level of consciousness, speech disorders, tremors and confusion.

[0082] 68. Autologous T cells expressing anti-CD19 CAR for use in a method for treating cancer according to any one of aspects 52 to 67.

[0083] 69. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a medicament for treating cancer according to any one of aspects 52 to 67.

[0084] 70. A method for treating cancer in a subject in need thereof, wherein the subject has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), further wherein a peripheral blood sample is collected from the subject after administration of the first T cell product, the method comprising (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.

[0085] 71. A method for treating cancer in a subject in need thereof, wherein the subject has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), and further wherein a peripheral blood sample is collected from the subject after administration of the first T cell product, the method comprising (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.

[0086] 72. A method according to aspect 70 or 71, wherein the first T cell product comprises CD4+ and CD8+ CART cells that have been prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and subsequent partial or complete depletion of circulating cancer cells.

[0087] 73. A method according to aspect 72, wherein the CD4+ and CD8+ T cells have been activated with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-2 and then transduced with a replication-defective viral vector encoding a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-ζ domains.

[0088] 74. The method according to any one of aspects 70 to 73, wherein the cancer is selected from the group consisting of mantle cell lymphoma (MCL), B-cell ALL, Waldenstrom's macroglobulinemia, Reye's transformation, Burkitt's lymphoma, and hairy cell leukemia.

[0089] 75. The method according to aspect 74, wherein the cancer is MCL.

[0090] 76. The method according to any one of aspects 70 to 75, wherein the blood sample is collected from the subject between day 5 and day 9 after administration of the first T cell product.

[0091] 77. The method according to aspect 76, wherein the blood sample is collected from the subject between day 6 and day 8 after administration of the first T cell product.

[0092] 78. The method according to aspect 77, wherein the blood sample is collected from the subject on day 7 after administration of the first T cell product.

[0093] 79. The method according to any one of aspects 70 to 75, wherein the blood sample is collected from the subject between day 12 and day 16 after administration of the first T cell product.

[0094] 80. The method of aspect 79, wherein the blood sample is collected from the subject between day 13 and day 15 after administration of the first T cell product.

[0095] 81. The method according to aspect 80, wherein the blood sample is collected from the subject on day 14 after administration of the first T cell product.

[0096] 82. The method of aspect 70, wherein the elevated CD8+CD27-CD28+ T cell levels in the subject are determined by comparison with other subjects who have received a comparable T cell product and whose peripheral blood samples were collected on the same day after administration of the T cell product.

[0097] 83. The method of aspect 71, wherein the elevated CD8+CCR7-CD45RA+CD27-CD28+ T cell levels in the subject are determined by comparison with other subjects who have received a comparable T cell product and whose peripheral blood samples are collected on the same day after administration of the T cell product.

[0098] 84. The method of any one of aspects 70 to 83, wherein the second T cell product is selected from the group consisting of an autologous CD19 / CD20 bicistronic T cell product and an allogeneic T cell product.

[0099] 85. A T cell product for use in a method for treating cancer according to any one of aspects 70 to 84.

[0100] 86. Use of a T cell product in the manufacture of a medicament for treating cancer according to any one of aspects 70 to 84.

[0101] 87. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising

[0102] (a) collecting a blood sample from the subject after administration of the first T cell product,

[0103] (b) measuring the level of CD8+CD27-CD28+ T cells in the blood sample, and

[0104] (c) prescribing a course of treatment 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, administering a second T cell product.

[0105] 88. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising

[0106] (a) collecting a blood sample from the subject after administration of the first T cell product,

[0107] (b) measuring the level of CD8+CCR7-CD45RA+CD27-CD28+ T cells in the blood sample, and

[0108] (c) prescribing a course of treatment 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, administering a second T cell product.

[0109] 89. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising

[0110] (a) collecting a blood sample from the subject after administration of the first T cell product,

[0111] (b) measuring the level of CD27+CD28- CD4+CD3+ T cells in said blood sample, and

[0112] (c) prescribing a course of treatment based on the level of CD27+CD28- CD4+ CD3+ T cells in said blood sample, wherein if the level of CD27+CD28- CD4+ CD3+ T cells is elevated, then not administering a second T cell product.

[0113] 90. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising

[0114] (a) collecting a blood sample from the subject after administration of the first T cell product,

[0115] (b) measuring the level of PD1+ CCR7+CD45RA- CD8+ CD3+ T cells in the blood sample, and

[0116] (c) prescribing a course of treatment 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, then not administering a second T cell product. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 The ZUMA-2 study design is shown. The superscripts are defined as follows: a Administer after leukapheresis and complete ≥5 days prior to initiation of conditioning chemotherapy; PET-CT required after bridging. b Bone marrow biopsy was performed at screening and, if positive, untested, or indeterminate, required biopsy to confirm CR. c After 3 months, only targeted AEs (neurological, hematological, infections, GVHD, autoimmune disorders, and secondary malignancies) were monitored and reported for 15 years after the initial anti-CD19 CAR T-cell infusion or until disease progression or initiation of subsequent anticancer agent therapy, whichever occurred first.

[0118] Figure 2 Patient response trends at the 24-month assessment are shown. Complete response (CR). Partial response (PR). DETAILED DESCRIPTION

[0119] Unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. For example, "The Concise Dictionary of Biomedicine and Molecular Biology", Juo, Pei-Show, 2nd edition, 2002, CRC Press; "The Dictionary of Cell and Molecular Biology", 3rd edition, 1999, Academic Press; and "Oxford Dictionary of Biochemistry and Molecular Biology", revised edition, 2000, Oxford University Press provides a general dictionary of many terms used in this application for technicians.

[0120] Units, prefixes and symbols are expressed in their SI accepted forms. Numerical ranges include numbers defining the range. The disclosure provided herein does not limit the various aspects of the present application, which can be referenced to this specification as a whole. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art related to the present disclosure. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd edition, 2001, CRC Press; "The Dictionary of Cell & Molecular Biology", 5th edition, 2013, Academic Press; and "The Oxford Dictionary of Biochemistry and Molecular Biology", edited by Cammack et al., 2nd edition, 2006, Oxford University Press provides a general dictionary of many terms used in the present disclosure for those skilled in the art.

[0121] The articles "a" and "an" refer to "one or more" of any stated or listed component.

[0122] The term "about" or "consisting essentially of" refers to a value or composition within an acceptable error range of a value or composition as determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice of the art, "about" or "consisting essentially of" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of" 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 (for 10%). With respect to biological systems or processes, the term may mean a value of up to an order of magnitude or up to 5 times. When certain values ​​or compositions are provided in the present application and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of" includes an acceptable error range for the value or composition. Any concentration range, percentage range, ratio range, or integer range includes the value of any integer within the stated range, and its fractions (such as one tenth and one hundredth of an integer) when appropriate, unless otherwise stated.

[0123] Unless otherwise specified or obvious from the context, as used herein, the term "or" is understood to be inclusive and covers both "or" and "and". The term "and / or" refers to each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein 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 cover each of the following: 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).

[0124] The terms "such as" and "ie" are used by way of example only, are not intended to be limiting, and are not to be construed as referring only to those items explicitly listed in the specification.

[0125] The terms “or more”, “at least”, “more than”, and the like, such as “at least one”, include but are not limited to 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 ,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90 , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 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, 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 than the value. Also included are any larger numbers or fractions therebetween. The term "not more than" includes every value less than the value. For example, "no more than 100 nucleotides" includes 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, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0126] The terms "plurality", "at least two", "two or more", "at least a second", etc. include but are not limited to 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, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 130, 131, 132, 133, 134, 135, 136, 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. Also included are any larger numbers or fractions therebetween.

[0127] Throughout the specification, the word "comprise" or variations such as "include" or "comprising" are understood to imply the inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. It should be understood that wherever aspects are described herein with the language "comprising," other similar aspects described as "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. 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 include materials other than those recited other than the impurities normally associated therewith"). The term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0128] Unless otherwise specified or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice of the art, "about" or "approximately" may mean within one or more than one standard deviation. "About" or "approximately" may mean a range of up to 10% (i.e., ± 10%). Therefore, "about" may be understood as being within 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 value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or processes, these terms may mean up to an order of magnitude or up to 5 times of a certain value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for the specific value or composition.

[0129] As described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range, and fractions thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise specified.

[0130] The terms "activation", "activated", etc. refer to a state of cells (including but not limited to immune cells (e.g., T cells)) that are sufficiently stimulated to induce detectable cell proliferation. Activation may be associated with induced cytokine production and detectable effector functions. The term "activated T cells" primarily refers to T cells that are undergoing cell division. T cell activation can 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 amplifying T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514 and PCT Publication No. WO2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. Typically, such methods involve contacting cells (such as T cells) with an activator, stimulatory agent, or co-stimulatory agent (such as anti-CD3 antibody and / or anti-CD28 antibody) that may be attached, coated, or bound to a bead or other surface in the presence of certain cytokines (such as IL-2, IL-7, and / or IL-15). Activators (such as anti-CD3 antibody and anti-CD28 antibody) attached to the same bead act as "surrogate" antigen presenting cells (APCs). An example is Dynabeads ® System, which is a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In one embodiment, T cells are activated and stimulated to proliferate with certain antibodies and / or cytokines using the methods described in U.S. Patents 6,040,177 and 5,827,642 and PCT Publication WO 2012 / 129514 (the contents of these patents are hereby incorporated by reference in their entirety).

[0131] The term "administration" (administration, Administering, etc.) refers to the physical introduction of a medicament into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for immune cells prepared by the methods disclosed herein include intravenous (iv or IV), intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). Parenteral routes of administration refer to modes of administration (usually by injection) other than enteral and topical administration, and include but are not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In one embodiment, immune cells (e.g., T cells) prepared by the methods of the present invention are administered by injection or infusion. Non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed once, twice, or multiple times over one or more extended time periods. Where one or more therapeutic agents (e.g., cells) are administered, the administration may be performed concomitantly or sequentially. Sequential administration includes administering one agent only after administration of another one or more agents has been completed.

[0132] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. In general, 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, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulins may be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. In the absence of explicit instructions, and unless otherwise indicated by the context, the term "antibody" also includes an antigen-binding fragment or antigen-binding portion of any of the aforementioned immunoglobulins, monovalent and divalent fragments or portions, and single-chain antibodies.

[0133] "Antigen binding molecules", "antibody fragments" and the like refer to any antibody portion that is smaller than the entire antibody. Antigen binding molecules may include antigen complementary determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed by antigen binding molecules. In one aspect, the CD19CAR construct comprises an anti-CD19 single-chain FV. A "single-chain Fv" or "scFv" antibody binding fragment comprises a heavy chain variable (V H ) domain and light chain variable (V L ) domains, wherein these domains are present in a single polypeptide chain. Generally speaking, the Fv polypeptide also includes V H Domain and V LThe polypeptide linker between the domains enables the scFv to form the desired structure for antigen binding. All antibody-related terms used herein have their usual meanings in the art and are well understood by those of ordinary skill in the art.

[0134] "Antigen" refers to any molecule that causes an immune response or can be bound by an antibody or antigen binding molecule. The immune response may involve antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule (including nearly all proteins or peptides) can be used as an antigen. Antigens may be endogenously expressed, i.e., expressed by genomic DNA, or may be recombinantly expressed. Antigens may be specific to a certain tissue (such as cancer cells), or they may be widely expressed. In addition, fragments of larger molecules may serve as antigens. In some embodiments, the antigen is a tumor antigen.

[0135] 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 effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand, or changes the binding ability of the ligand by indirect means (e.g., structural or energetic changes in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it binds from performing a biological function.

[0136] The term "autologous" refers to any material derived from the same individual and later reintroduced into the individual. For example, the engineered autologous cell therapy methods described herein involve collecting lymphocytes from an individual (such as a donor or patient), then engineering them to express a CAR construct, and then administering them back to the same individual.

[0137] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0138] The term "bridging therapy" refers to treatment given between apheresis / leukapheresis and the start of lymphodepleting / conditioning chemotherapy.

[0139] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also be transferred to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors at various stages. In one embodiment, the cancer or tumor is in stage 0, so that, for example, the cancer or tumor is in very early development and has not yet metastasized. In another embodiment, the cancer or tumor is in stage I, so that, for example, the size of the cancer or tumor is relatively small, has not spread to nearby tissues, and has not yet metastasized. In other embodiments, the cancer or tumor is in stage II or stage III, so that, for example, the cancer or tumor is greater than stage 0 or stage I, and it has grown into adjacent tissues, but it has not yet metastasized, except potentially lymph nodes. In additional embodiments, the cancer or tumor is in stage IV, so that, for example, the cancer or tumor has metastasized. Stage IV can also be referred to as advanced or metastatic cancer.

[0140] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as, but is not limited to, a reduction in tumor volume, inhibition of tumor growth, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number / extent of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumorigenesis, such as vaccines.

[0141] The term "progression-free survival" (PFS) refers to the time 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 due to any cause. The term "disease progression" can be assessed by measurement of malignant lesions on radiographs or other methods and should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms can be reported as the primary tumor type (e.g., DLBCL). The term "duration of response" (DOR) refers to the time period between the subject's first objective response 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 time from the date of treatment to the date of death.

[0142] "Cytokine" refers to a non-antibody protein released by immune cells (including 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 therapy. In other embodiments, those cytokines secreted in response to therapy can indicate or prompt effective therapy. In one embodiment, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. As used herein, "cytokine" refers to a protein released by a cell population that acts on another cell as an intercellular mediator. Cytokines can be expressed endogenously 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 immune responses. Cytokines can induce various responses in receptor cells. Cytokines can include steady-state cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, steady-state cytokines including interleukin (IL) 7 and IL-15 promote immune cell survival and proliferation, and proinflammatory 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)γ. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF) 2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell 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).

[0143] A "chemokine" is a 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 chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymic activation-regulated chemokine (TARC or CCL17).

[0144] "Therapeutically effective amount", "therapeutically effective dose", etc. refers to the amount of cells (such as immune cells or engineered T cells) produced by the methods of the present invention (producing T cell products) and protecting or treating subjects from the onset of disease or promoting disease regression (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease asymptomatic periods, and / or prevention of damage or disability caused by disease affliction) when used alone or in combination with another therapeutic agent. The ability to promote disease regression can be assessed using a variety of methods known to technicians (such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of agents in in vitro assays). In some embodiments, donor T cells for T cell therapy are obtained from patients (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from subjects who are not patients. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can 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 In another embodiment, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or about 9×10 7 In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 1×10 cells / kg body weight. 6 With about 2×10 6 The number of CAR-positive live T cells ranges from about 1×10 8 In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 0.4×10 8 With about 2×10 8 In some embodiments, the therapeutically effective amount of CAR-positive live T cells is about 0.4×10 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 about 2.0×10 8 CAR-positive live T cells.

[0145] 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 (toxic to cells) lymphocytes that represent a major component of the innate immune system. NK cells reject tumors and cells infected by viruses through apoptosis or programmed cell death. They are called "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) distinguish themselves from other lymphocyte types. The thymus is a specialized organ of the immune system that is primarily responsible for the maturation of T cells.

[0146] There are several types of "immune cells", including but 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 (TIL), bone marrow-derived suppressor cells (MDSC) and dendritic cells. The term also includes the precursors of these immune cells. Hematopoietic stem cells and / or progenitor cells can be derived from bone marrow, umbilical cord blood, adult peripheral blood after cytokine mobilization, etc. by methods known in the art. Some precursor cells are those cells that can differentiate into lymphoid lineages (e.g., hematopoietic stem cells or progenitor cells of lymphoid lineages). Additional examples of immune cells that can be used for immunotherapy are described in U.S. Publication No. 20180273601, which is incorporated herein by reference in its entirety.

[0147] There are several types of T cells, namely: helper T cells (e.g., CD4+ cells, effector T EFF cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory T SCM Central memory T cells (i.e., naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and display many functional attributes that are characteristic of memory cells); (ii) central memory T cells CM Cells express L-selectin and are CCR7 + and CD45RO + They secrete IL-2 but not IFNγ or IL-4, whereas (iii) effector memory T EM cells do not express L-selectin or CCR7 but do express CD45RO and produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4 + CD25 + T cells are classified into three types: regulatory T cells, natural killer T cells (NKT), and gamma delta T cells. T cells found in tumors are called "tumor infiltrating lymphocytes" (TIL). On the other hand, B cells play a major role in humoral immunity (involving antibodies). They make antibodies and antigens and function 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 from which they are named.

[0148] "Naive" T cells are mature T cells that remain undifferentiated. After positive and negative selection in the thymus, T cells are formed as CD4 + or CD8 + Naive T cells appear. In their naive state, T cells express L-selectin (CD62L + ), IL-7 receptor α (IL-7R-α) and CD132, but they do not express CD25, CD44, CD69 or CD45RO. As used herein, "immature" can also refer to T cells that exhibit naive or immature T cells (such as T SCM Cells or T CM- - For example, immature T cells may express L-selectin (CD62L + ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive or immature T cells can interact with terminally differentiated effector T cells (such as T EM Cells and T EFF cells) in contrast.

[0149] As mentioned 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 method of the present application for preparing T cells under certain oxygen and / or pressure conditions will increase one or more T cell functions, thereby making T cells more suitable and / or more effective for therapeutic purposes. In some embodiments, compared with those under the conditions lacking certain oxygen and / or pressure, the T cells prepared according to the inventive method have increased T cell function. In other embodiments, compared with T cells cultured under the conditions lacking certain oxygen and / or pressure, the T cells prepared according to the inventive method will have increased T cell proliferation. In additional embodiments, compared with T cells cultured under the conditions lacking certain oxygen and / or pressure, the T cells prepared according to the inventive method have increased T cell activity. In other embodiments, compared with T cells cultured under the conditions lacking certain oxygen and / or pressure, the T cells prepared according to the inventive method have increased cytolytic activity.

[0150] The term "proliferation" (proliferation or proliferating, etc.) of cells refers to the ability of cells to grow digitally by cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation may occur in vitro (e.g., during T cell culture) or in vivo (e.g., after administering immune cell therapy (e.g., T cell therapy)). Cell proliferation can be measured or determined by methods described herein or known in the art. For example, cell proliferation can be measured or determined by viable cell density (VCD) or total viable cells (TVC). VCD or TVC can be theoretical (taking aliquots or samples from the culture at a certain time point to determine the number of cells, and then multiplying the number of cells by the culture volume at the beginning of the study) or actual (taking aliquots or samples from the culture at a certain time point to determine the number of cells, and then multiplying the number of cells by the actual culture volume at a certain 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 (such as INFγ, IL-2 and / or TNFα). In other embodiments, T cell activity includes the production of one or more cytokines selected from interferon gamma (IFNγ or IFN-γ), tissue necrosis factor alpha (TNFα or IFNα), and both. The terms "cytolytic activity", "cytotoxicity", etc. refer to the ability of T cells to destroy target cells. In one embodiment, the target cell is a cancer cell, such as a tumor cell. In other embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cell expresses a target antigen.

[0151] The terms "genetically engineered", "gene editing" or "engineered" refer to methods of modifying the genome of a cell, including but not limited to deleting a coding region or a non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In one embodiment, the modified cell is a lymphocyte (e.g., a T cell) obtainable from a patient or a donor. The cell can be modified to express an exogenous construct incorporated into the cell's genome, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0152] The terms "transduction" and "transduced" refer to the process of introducing exogenous DNA into cells by viral vectors (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0153] The chimeric antigen receptor (CAR or CAR-T) and T cell receptor (TCR) of the present application are genetically engineered receptors. According to techniques known in the art, these engineered receptors can be easily inserted into and expressed by immune cells including T cells. Using CAR, a single receptor can be programmed to both recognize a specific antigen and activate immune cells to attack and destroy cells carrying or expressing the antigen when binding to the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. In one embodiment, the cell prepared according to the present application is a cell having a chimeric antigen receptor (CAR) or T cell receptor comprising an antigen binding molecule, a costimulatory domain, and an activation domain. The costimulatory domain may include an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain includes a hinge or a truncated hinge domain.

[0154] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that result in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the body of a vertebrate.

[0155] The term "immunotherapy" (immune therapy, etc.) refers to the treatment of a subject suffering from a disease or at risk of developing a disease or suffering from a recurrence of a disease by a method that includes inducing, enhancing, suppressing or otherwise changing an immune response. Examples of immunotherapy include, but are not limited to, T cell and NK cell therapy. T cell therapy 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 recognize that the methods for preparing immune cells disclosed herein will enhance the efficacy of any cancer or transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409; U.S. Patent Nos. 7,741,465; 6,319,494 and 5,728,388; and PCT Publication No. WO 2008 / 081035, which are incorporated by reference in their entirety.

[0156] The term “engineered autologous cell therapy” (abbreviated as “eACT”) ™”, also known as adoptive cell transfer) is a process by which a patient’s own T cells are collected and subsequently genetically engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable region fragment (scFv) specific for a certain tumor antigen, which is linked to an intracellular signaling moiety comprising a co-stimulatory domain and an activation domain.A "costimulatory domain" can be a signaling region derived from, 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 co-stimulator (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig α (CD79a), DAP-10, Fc γ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8, CD8α, CD8β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDllb, ITGAX, CDl lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof. The activation domain can be derived from, for example, CD3, such as CD3ζ, ε, δ, γ, etc. In one embodiment, the CAR is designed to have two, three, four or more co-stimulatory domains.CARscFv can be designed to target, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage (including all normal B cells and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL). Exemplary CAR+ T cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are hereby incorporated by reference in their entirety.

[0157] As used herein, "co-stimulatory signal" refers to a signal that, in conjunction with a primary signal such as TCR / CD3 ligation, elicits a T cell response such as, but not limited to, proliferation and / or up- or down-regulation of key molecules.

[0158] As used herein, "costimulatory ligands" include molecules on antigen presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of costimulatory ligands provides signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.). Costimulatory ligands induce signals in addition to the primary signals provided by stimulatory molecules, for example, through the binding of T cell receptor (TCR) / CD3 complexes to major histocompatibility complex (MHC) molecules loaded with peptides. Co-stimulatory ligands may include, but are not limited to, 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, herpes virus 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 beta 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. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to 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).

[0159] "Costimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as but not limited to proliferation. Co-stimulatory molecules include, but are not limited to, "Co-stimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as but not limited to proliferation. Co-stimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD 45. CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD1 9a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (α; β; δ; ε; γ; ζ), CD30, CD37 , CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8α, CD8β, 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 la / CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncations or combinations thereof.

[0160] In some aspects, the cells of the present application can be obtained by T cells obtained from a subject. In one aspect, T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. A variety of techniques known to technicians (such as FICOLL ™ T cells are obtained from a blood unit collected from a subject by separation and / or apheresis. In some aspects, the cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing. In some aspects, the cells are washed with any solution (e.g., a solution with a neutralized pH or PBS) or culture medium. It should be understood that a washing step can be used, such as by using a semi-automatic flow-through centrifuge, such as a Cobe ™ 2991 Cell Processor, Baxter CytoMate ™ Etc. In some aspects, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In some aspects, unwanted components of the blood apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is hereby incorporated by reference in its entirety.

[0161] In some embodiments, the monocytes are depleted by lysing red blood cells (e.g., by using a PERCOLL ™ Gradient centrifugation) is used to separate T cells from PBMC. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+ and CD45RO+T cells, are further separated by positive or negative selection techniques known in the art. For example, a combination of antibodies for surface markers specific to negatively selected cells can be used to complete the enrichment of T cell populations by negative selection. In some embodiments, cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry can be used, which uses a mixture of monoclonal antibodies for cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, a monoclonal antibody mixture generally includes antibodies for CD8, CD11b, CD14, CD16, CD20 and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to separate the cell population of interest used in the present disclosure.

[0162] In one embodiment, CD3+ T cells are isolated from PBMCs using Dynabeads coated with anti-CD3 antibodies. CD8+ and CD4+ T cells are further isolated separately by positive selection using CD8 microbeads (e.g., Miltenyi Biotec, Germany) or CD4 microbeads (e.g., Miltenyi Biotec, Germany).

[0163] In some embodiments, PBMCs are used directly for genetic modification of immune cells (such as CARs) using methods as described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated, and cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subsets before or after genetic modification and / or expansion.

[0164] One or more immune cells described herein may be obtained from any source, including, for example, human donors. The donor may be a subject (i.e., an autologous donor) who needs anti-cancer treatment (e.g., treatment with immune cells produced by the methods described herein), or may be an individual (i.e., an allogeneic donor) who donates a lymphocyte sample, which will be used to treat a different individual or cancer patient after producing a cell population produced by the methods described herein. Immune cells may be differentiated from a hematopoietic stem cell population in vitro or immune cells may be obtained from a donor. Immune cell populations may be obtained from donors by any suitable method used in the art. For example, lymphocyte populations may be obtained by any suitable in vitro method, venipuncture, or other blood collection method, by which a blood sample with or without lymphocytes is obtained. Lymphocyte populations are obtained by apheresis. One or more immune cells may be collected from any tissue (including, but not limited to, a tumor) containing one or more immune cells. A tumor or a portion thereof is collected from a subject, and one or more immune cells are separated from the tumor tissue. Any T cell may be used in the methods disclosed herein, including any immune cell suitable for T cell therapy. For example, one or more cells that can be used in the present application can be selected from the group consisting of: tumor infiltrating lymphocytes (TIL), 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 from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. A variety of techniques known to technicians (such as FICOLL ™T cells are obtained from a blood unit collected from a subject by separation and / or apheresis. T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system, which replicates the human thymus environment to support efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication Nos. 2013 / 0287748, PCT Publication Nos. WO2015 / 120096, and WO2017 / 070395, all of which are incorporated herein by reference in their entirety for the purpose of describing these methods. In one embodiment, the T cells are tumor infiltrating leukocytes. In certain embodiments, the one or more T cells express CD8, for example, CD8 + In other embodiments, the one or more T cells express CD4, e.g., are CD4 + T cells. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, PCT Publication No. WO2015 / 120096, and WO2017 / 070395, all of which are incorporated herein by reference in their entirety for the purpose of describing these methods.

[0165] Immune cells and their precursor cells can be separated by available methods (see, for example, Rowland-Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York, 1999). The sources of immune cells or their precursor 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. In addition, positive selection methods can separate or enrich the desired immune cells or their precursor cells, or a combination of positive and negative selection methods can be used. Monoclonal antibodies (MAbs) can be used to identify markers associated with certain cell lineages and / or differentiation stages of both positive and negative selection. If certain types of cells are to be isolated (e.g., certain types of T cells), various cell surface markers or combinations of markers (including, but not limited to, CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells), etc.) can be used to isolate the cells, as is well known in the art (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa NJ, USA (2000); De Libero, T Cell Protocols, Volume 514 in Methods in Molecular Biology, Humana Press, Totowa NJ, USA (2009)).

[0166] PBMC can be used directly for genetic modification of immune cells (such as CAR). After separating PBMC, T lymphocytes are further separated, and before or after genetic modification and / or amplification, both cytotoxic and helper T lymphocytes are sorted into immature, memory and effector T cell subsets. In one embodiment, by identifying cell surface antigens associated with each of these types of CD8+ cells, CD8+ cells can be further sorted into immature, central memory and effector cells. In other embodiments, the expression of phenotypic markers of 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 a certain embodiment, effector T cells are negative for CCR7, CD28, CD62L and CD127 and are positive for granzyme B and perforin. In additional embodiments, CD4+ T cells can be further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

[0167] The methods described herein further include enriching or preparing a population of immune cells obtained from a donor between harvesting from the donor and exposing one or more cells obtained from the donor subject. Enriching a population of immune cells (e.g., one or more T cells) can be accomplished by any suitable separation method, including but not limited to using a separation medium (e.g., FICOLL-PAQUE TM ROSETTESEP ™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Catalog No. 0850494X, etc.), cell size, shape, or density separation by filtration or panning, immunomagnetic separation (e.g., magnetic activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

[0168] In one embodiment, 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 application also provides a composition or a preparation comprising a pharmaceutically acceptable carrier, a diluent, a solubilizer, an emulsifier, a preservative and / or an adjuvant. In certain embodiments, the composition or preparation comprises an excipient. The term composition or preparation is used interchangeably herein. The term 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, for inhalation or for delivery through the digestive tract, such as oral administration. The composition can be prepared by a person skilled in the art by known methods. The composition is maintained at a physiological pH or a slightly lower pH using a buffer, typically in the pH range of about 5 to about 8. When considering parenteral administration, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution in a pharmaceutically acceptable solvent, the aqueous solution comprising the composition described herein, with or without an additional therapeutic agent. For example, the vehicle for parenteral injection is sterile distilled water, in which the compositions described herein are formulated as sterile isotonic solutions that are appropriately preserved with or without at least one additional therapeutic agent. Preparation involves formulating the desired agent with a polymer compound (such as polylactic acid or polyglycolic acid), beads or liposomes to provide controlled or sustained release of the product, which is then delivered by depot injection. In addition, implantable drug delivery devices can be used to introduce the desired therapeutic agent.

[0169] In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject who is not a patient. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can 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 In another embodiment, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2×10 6 cells / kg, about 3×106 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or about 9×10 7 In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 1×10 cells / kg body weight. 6 With about 2×10 6 The number of CAR-positive live T cells ranges from about 1×10 8 The maximum dose of viable CAR-positive T cells.

[0170] As used herein, "patient" includes any person suffering from a disease or disorder, including cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers to a subject whose cells are obtained for further in vitro engineering. The donor subject can be a cancer patient (i.e., an autologous donor) who will be treated with a cell population produced by the methods described herein, or can be an individual (i.e., an allogeneic donor) who donates a lymphocyte sample that will be used to treat a different individual or cancer patient after generating a cell population produced by the methods described herein. Those subjects who receive cells prepared by the methods of the present invention can be referred to as "recipient subjects."

[0171] The term "stimulation" (stimulation or stimulating, etc.) refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell (e.g., a T cell receptor (TCR) / CD3 complex) that specifically binds to a cognate stimulatory ligand presented on an antigen presenting cell. A "stimulatory ligand" is a ligand that can specifically bind to a stimulatory molecule on a T cell when presented on an antigen presenting cell (e.g., an artificial antigen presenting cell (aAPC), a dendritic cell, a B cell, etc.), thereby mediating a primary response of the T cell (including but not limited to activation, an initial immune response, proliferation, etc.). Stimulatory ligands include but are not limited to MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. As used herein, "activated" or "active" refers to a T cell that has been stimulated. Active 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.

[0172] The term "exogenous activation material" refers to any activating substance derived from an external source. For example, exogenous anti-CD3 antibodies, anti-CD28 antibodies, IL-2, exogenous IL-7, or exogenous IL-15 may be commercially available or recombinantly produced. When added to or in contact with one or more T cells, "exogenous IL-2," "exogenous IL-7," or "exogenous IL-15" means that the T cells do not produce such IL-2, IL-7, and / or IL-15. T cells prior to mixing with "exogenous" IL-2, IL-7, or IL-15 may contain traces (i.e., endogenous "exogenous" IL-2, IL-7, or IL-15) produced by the T cells or separated from the subject together with the T cells. The one or more T cells described herein can be contacted with exogenous anti-CD3 antibodies, anti-CD28 antibodies, "exogenous" IL-2, IL-7 and / or IL-15 by any means known in the art, including adding isolated "exogenous" IL-2, IL-7 and / or IL-15 to the culture medium, including anti-CD3 antibodies, anti-CD28 antibodies, "exogenous" IL-2, IL-7 and / or IL-15 in the culture medium, or expressing "exogenous" IL-2, IL-7 and / or IL-15 by one or more cells in the culture other than the one or more T cells (such as by a feeder layer).

[0173] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In one embodiment, the in vitro cell comprises a T cell.

[0174] The term "persistence" refers to, for example, one or more transplanted immune cells or their progenitor cells (e.g., differentiated or mature T cells) administered to a subject, and the ability to retain a period of time in the subject at a detectable level. As used herein, increasing the persistence of one or more transplanted immune cells or their progenitor cells (e.g., differentiated or mature T cells) refers to increasing the amount of time that transplanted immune cells can be detected in a subject after administration. For example, the in vivo persistence of one or more transplanted immune cells can be increased by at least about 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. Such, the in vivo persistence of one or more transplanted immune cells can be increased 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 of the invention disclosed herein.

[0175] The terms "reduce" and "reduced" are used interchangeably herein and indicate any change that is less than the original value. "Reduce" and "reduced" are relative terms that require a comparison between before and after a measurement. "Reduce" and "reduced" include complete depletion. As used herein, the term "regulating" T cell maturation refers to controlling the maturation and / or differentiation of one or more cells (such as T cells) using any intervention described herein. For example, regulating refers to inactivating, delaying or inhibiting T cell maturation. In another example, regulating refers to accelerating or promoting T cell maturation. The term "delaying or inhibiting T cell maturation" refers to maintaining one or more T cells in an immature or undifferentiated state. For example, "delaying or inhibiting T cell maturation" can refer to maintaining T cells in a naive or T cell state. CM status, and progress to T EM or T EFF- In addition, "delaying or inhibiting T cell maturation" can refer to increasing or enriching immature or undifferentiated T cells (e.g., naive T cells and / or T CM- -The state of the T cells (e.g., as 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 the 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 can indicate less mature, undifferentiated T cells.

[0176] "Treatment" or "treatment" of a subject / patient refers to any type of intervention or process performed on a subject / patient, or administration of one or more T cells prepared by the present application to a subject / patient, with the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with a disease. In one aspect, "treatment" or "treatment" includes partial remission. In another aspect, "treatment" or "treatment" includes complete remission.

[0177] Various aspects of the application are described in further detail in the following subsections.

[0178] Patients with B-cell malignancies that have high levels of circulating tumor cells expressing CD19 represent a population with very high unmet needs. For example, mantle cell lymphoma (MCL) is challenging to treat in the relapsed or refractory setting and remains incurable. There is no standard of care for second-line and higher-level chemotherapy. Treatment options include cytotoxic chemotherapy, proteasome inhibitors, immunomodulatory drugs, tyrosine kinase inhibitors, and stem cell transplantation (both autologous [ASCT] and allogeneic stem cell transplantation [allo-SCT]). The choice of regimen is influenced by prior therapy, comorbidities, and tumor chemosensitivity. Despite high initial response rates observed with Bruton's tyrosine kinase inhibitors (BTK inhibitors), the majority of patients will eventually develop progressive disease. New treatment strategies are needed to improve the poor prognosis of patients with r / r MCL whose disease has not been effectively controlled with chemoimmunotherapy, stem cell transplantation, and BTK inhibitors.

[0179] The anti-CD19 CAR T cell therapy or product used in CD19 CAR-T can be made from the patient's own T cells, and the tumor cells expressing CD19 in the final product are minimized by leukapheresis suitable for B cell malignancies with circulating tumor cell load. T cells of leukapheresis products can be enriched by selecting CD4+ / CD8+ T cells, activated with anti-CD3 antibodies and anti-CD28 antibodies, and / or transduced with viral vectors containing anti-CD19 CAR genes. More details of the method can be found in PCT / US2015 / 014520 published as WO2015 / 120096 and PCT / US2016 / 057983 published as WO2017 / 070395. In one embodiment, the cells are not treated with AKT inhibitors, IL-7 and IL-15. These engineered T cells can be propagated to produce a sufficient number of cells to achieve a therapeutic effect. Such procedures remove malignant and normal B cells expressing CD19, which can reduce the activation, expansion, and exhaustion of anti-CD19 CAR T cells.

[0180] The activation, transduction and / or expansion of immune cells can be performed at any suitable time that allows (i) the production of a sufficient number of cells in the engineered immune cell population for at least one dose administered to a patient, (ii) the production of an engineered immune cell population with a favorable ratio of immature cells compared to typical longer processes, or (iii) both (i) and (ii). The appropriate time may involve several parameters, including the population of one or more cells, the cell surface receptors expressed by the immune cells, the vector used, the dosage required for the therapeutic effect, and / or other variables. The activation time can be 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. Compared to amplification methods known in the art, the activation time according to the method of the present application will be reduced. In some embodiments, the activation time can 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 75% shorter. In addition, the amplification time can be 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more than 21 days. Compared with amplification methods known in the art, the amplification time according to the method of the application will be reduced. For example, the expansion time can 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 can be 75% shorter. In one embodiment, the time for cell expansion is about 3 days, and the time from enrichment of the cell population producing the engineered immune cell is about 6 days.

[0181] The delay 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, the delay 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). The one or more biomarkers are selected from the group consisting of: L-selectin (CD62L +), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof. In certain aspects, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be detected by detecting L-selectin (CD62L + ), IL-7Rα and CD132. Those skilled in the art will recognize that although the methods of the present invention can increase the relative proportion of immature and undifferentiated T cells or DC cells in the collected cell population, some mature and differentiated cells may still be present. Therefore, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by calculating the total percentage of immature and undifferentiated cells in the cell population before and after exposing one or more cells obtained from the donor subject to hypoxic culture conditions with or without a pressure higher than atmospheric pressure. The methods disclosed herein can increase the percentage of immature and undifferentiated T cells in a T cell population.

[0182] The methods described herein also include stimulating a cell population (such as lymphocytes) with one or more T cell stimulants to produce an activated T cell population under suitable conditions. Any combination of one or more suitable T cell stimulants can be used to produce an activated T cell population, including but not limited to antibodies or functional fragments thereof targeting T cell stimulation or co-stimulatory 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., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM) or natural ligands of T cell stimulation or co-stimulatory molecules.

[0183] Suitable conditions for stimulating or activating immune cell populations as described herein also include temperature, for a certain amount of time, and / or in the presence of CO2 levels. The temperature of stimulation can be about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34°C-38°C, about 35°C-37°C, about 36°C-38°C, about 36°C-37°C, or about 37°C.

[0184] Another condition for stimulating or activating an immune cell population as described herein can also include the time of stimulation or activation. The time of stimulation is about 24-72 hours, about 24-36 hours, about 30–42 hours, about 36–48 hours, about 40–52 hours, about 42–54 hours, about 44–56 hours, about 46–58 hours, about 48–60 hours, about 54–66 hours, or about 60–72 hours, about 44–52 hours, about 40–44 hours, about 40–48 hours, about 40–52 hours, or about 40–56 hours. In one embodiment, the time of stimulation is about 48 hours or at least about 48 hours.

[0185] Other conditions for stimulating or activating immune cell populations as described herein may also include CO2 levels. The CO2 level for stimulation is 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, about 4.5-5.5% CO2. In one embodiment, the CO2 level for stimulation is about 5% CO2.

[0186] Conditions for stimulating or activating immune cell populations may also include temperature, a certain amount of time for stimulation, and / or any combination in the presence of CO2 levels. For example, the step of stimulating immune cell populations may include stimulating immune cell populations with one or more immune cell stimulants for about 44-52 hours at a temperature of about 36-38°C and in the presence of a CO2 level of about 4.5%–5.5% CO2. One or more immune cells of the present application may be administered to a subject for immunization or cell therapy. Therefore, one or more immune cells may be collected from a subject in need of immunization or cell therapy. Once collected, one or more immune cells may be processed for any suitable time period before being administered to a subject.

[0187] The concentration, amount or population of lymphocytes or products produced by the methods herein is about 1.0 - 10.0 × 10 6 In some aspects, the concentration is about 1.0 – 2.0×10 6 cells / mL, about 1.0 – 3.0×10 6 cells / mL, about 1.0 – 4.0×10 6 cells / mL, about 1.0 – 5.0×10 6 cells / mL, about 1.0 – 6.0×10 6 cells / mL, about 1.0 – 7.0×10 6cells / mL, about 1.0 – 8.0×10 6 cells / mL, 1.0 – 9.0×10 6 cells / mL, about 1.0 – 10.0×10 6 cells / mL, about 1.0 – 1.2×10 6 cells / mL, about 1.0 – 1.4×10 6 cells / mL, about 1.0 – 1.6×10 6 cells / mL, about 1.0 – 1.8×10 6 cells / mL, about 1.0 – 2.0×10 6 cells / mL, at least about 1.0×10 6 cells / mL, at least about 1.1×10 6 cells / mL, at least about 1.2×10 6 cells / mL, at least about 1.3×10 6 cells / mL, at least about 1.4×10 6 cells / mL, at least about 1.5×10 6 cells / mL, at least about 1.6×10 6 cells / mL, at least about 1.7×10 6 cells / mL, at least about 1.8×10 6 cells / mL, at least about 1.9×10 6 cells / mL, at least about 2.0×10 6 cells / mL, at least about 4.0×10 6 cells / mL, at least about 6.0×10 6 cells / mL, at least about 8.0×10 6 cells / mL or at least about 10.0×10 6 cells / mL.

[0188] An anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody can be used in conjunction with or independently of the step of stimulating a lymphocyte population by exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without a pressure higher than atmospheric pressure. Any soluble or fixed anti-CD2 antibody, anti-CD3 antibody, and / or anti-CD28 antibody or a functional fragment thereof (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)) can be used. In some aspects, antibodies can be purchased commercially from suppliers known in the art, including, but not limited to, German Tianmei Biotech, BD Biosciences (e.g., 1 mg / mL pure MACSGMP CD3, part number 170-076-116) and eBioscience. In addition, those skilled in the art will understand how to produce anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some aspects, one or more T cell stimulants used according to the step of stimulating lymphocyte populations include antibodies or their functional fragments targeting T cell stimulation or costimulatory molecules in the presence of T cell cytokines. In one embodiment, the one or more T cell stimulants include anti-CD3 antibodies and IL-2. In certain embodiments, the T cell stimulant includes an anti-CD3 antibody at a concentration of 50ng / mL. The concentration of anti-CD3 antibodies is about 20ng / mL-100ng / mL, about 20ng / mL, about 30ng / mL, about 40ng / mL, about 50ng / mL, about 60ng / mL, about 70ng / mL, about 80ng / mL, about 90ng / mL or about 100ng / mL. In alternative aspects, T cell activation is not required.

[0189] The method described herein also includes transducing the activated immune cell population with a viral vector containing a nucleic acid molecule encoding a cell surface receptor using a single or multiple viral transduction cycle to produce a transduced immune cell population. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that can be used according to the transduction step can be any idiosyncratic or bidirectional viral vector, including but not limited to a recombinant retroviral vector, a recombinant lentiviral vector, a recombinant adenoviral vector, and a recombinant adeno-associated virus (AAV) vector. The method also includes transducing the one or more immune cells with a retrovirus. In one aspect, the viral vector used to transduce the activated immune cell population is a MSGV1 γ retroviral vector. In one embodiment, the viral vector used to transduce the activated immune cell population is a PG13-CD19-H3 vector described by the following document: Kochenderfer, Journal of Immunotherapy (J. Immunother.) 32 (7): 689–702 (2009). According to one aspect of this aspect, the viral vector is grown in a suspension culture in a culture medium specific for viral vector manufacturing (referred to herein as a viral vector inoculum). According to the methods described herein, any suitable growth medium and / or supplement for growing viral vectors can be used in the viral vector inoculum. According to some aspects, the viral vector inoculum is then added to the serum-free medium described below during the transduction step. In some aspects, the one or more immune cells can 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 can bind to an antigen on the surface of a target cell (e.g., 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 a pressure higher than atmospheric pressure, the conditions for transducing an activated immune cell population as described herein may include a specific time, at a specific temperature, and / or in the presence of a specific CO2 level. The transduction temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34°C-38°C, about 35°C-37°C, about 36°C-38°C, about 36°C-37°C. In one embodiment, the transduction temperature is about 37°C. The predetermined transduction temperature may be about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., or about 39° C., about 34° C.-39° C., about 35° C.-37° C. In one embodiment, the predetermined transduction temperature may be about 36° C.-38° C., about 36° C.-37° C., or about 37° C.The transduction time is about 12 hours to 36 hours, about 12 hours to 16 hours, about 12 hours to 20 hours, about 12 hours to 24 hours, about 12 hours to 28 hours, about 12 hours to 32 hours, about 20 hours, or at least about 20 hours, about 16 hours to 24 hours, about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours. The CO2 level used for transduction is 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%, about 10.0% CO2, about 3-7% CO2, about 4-6% CO2, about 4.5-5.5% CO2, or about 5% CO2.

[0190] Transduction of an activated immune cell population as described herein can be performed in any combination over a period of time, at a temperature, and / or in the presence of a specific CO2 level: a temperature of about 36°C-38°C, for an amount of time of about 16 hours-24 hours, and in the presence of a CO2 level of about 4.5%-5.5% CO2. Immune cells can be prepared by any of the methods of the present application in combination with any manufacturing method for preparing T cells for immunotherapy, including but not limited to those described in PCT publications WO2015 / 120096 and WO2017 / 070395, which are incorporated herein by reference in their entirety for the purpose of describing these methods; for preparing Akilencel or Yescarta ® Any and all methods for preparing tesalencyclidine / Kymriah ™ Any and all methods for preparing "ready-made" T cells for immunotherapy; any and all methods for preparing lymphocytes for administration to humans. The manufacturing method can be adapted to remove circulating tumor cells from cells obtained from a patient.

[0191] CAR-T cells can be engineered to express other molecules, and these CAR-T cells can be any of the following exemplary types or other types available in the art: first, second, third, fourth, fifth or more CAR-T cells; Armored CAR-T cells, athletic 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. In one aspect, the T cell is an autologous T cell. In one aspect, the T cell is an autologous stem cell (for autologous stem cell therapy or ASCT). In one aspect, the T cell is a non-autologous T cell.

[0192] Cells (such as immune cells or T cells) are genetically modified using known methods after isolation or selection, or activated and / or expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified. Immune cells (e.g., T cells) are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and activated and / or expanded in vitro. Methods for activating and expanding T cells can be found in U.S. Patents Nos. 6,905,874, 6,867,041, and 6,797,514 and PCT Publication No. WO 2012 / 079000, which are hereby incorporated by reference. In general, such methods can include contacting PBMCs or isolated T cells with stimulators and co-stimulators (such as anti-CD3 antibodies and / or anti-CD28 antibodies) that can be attached to beads or other surfaces in a culture medium with certain cytokines (such as IL-2). Dynabeads can be administered ® The invention relates to a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. T cells can be activated and stimulated to proliferate with appropriate feeder cells, antibodies and / or cytokines, as described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514, which are hereby incorporated by reference in their entirety.

[0193] The cell surface receptor expressed by the engineered immune cell can be any antigen or molecule targeted by a CAR, such as an anti-CD19 CAR, a FMC63-28Z CAR, or a FMC63-CD828BBZ CAR (Kochenderfer et al., J Immunother. 2009, 32(7): 689; Locke et al., Blood, 2010, 116(20): 4099, the subject matter of both documents is hereby incorporated by reference. In certain aspects, the predetermined dose of engineered immune cells can be more than about 1 million to less than about 3 million transduced engineered T cells / kg. In one embodiment, the predetermined dose of engineered T cells can be more than about 1 million to about 2 million transduced engineered T cells / kilogram of body weight (cells / kg). The predetermined dose of engineered T cells can be more than 1 million to about 2 million, at least about 2 million to less than about 3 million transduced Engineered T cells / kilogram body weight (cells / kg). In one embodiment, a predetermined dose of engineered T cells can be about 2 million transduced engineered T cells / kg. In another embodiment, a predetermined dose of engineered T cells can be at least about 2 million transduced engineered T cells / kg. Examples of predetermined doses of engineered T cells can 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 engineered T cells / kg.

[0194] Methods described herein include increasing or enriching the immune cell group of one or more transductions for a period of time to produce an engineered immune cell group. The expansion time can be any suitable time, and any suitable time allows (i) to produce a sufficient number of cells in the engineered immune cell group for at least one dosage applied to the patient, (ii) to produce an engineered immune cell group with a favorable ratio of young cells compared to a typical long process, or (iii) (i) and (ii) both. The time will depend on the cell surface receptors expressed by the immune cells, the carrier used, the dosage required for the therapeutic effect and other variables. The predetermined expansion time can be 0 days, 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more than 21 days. In one embodiment, compared with methods known in the art, the expansion time of the inventive method is reduced. For example, the predetermined expansion time can 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 can be 75% shorter. In one example, the expansion time is about 3 days, and the time from enrichment of the lymphocyte population that produces the engineered immune cell is about 6 days.

[0195] Conditions for expanding the transduced immune cell population may include temperature and / or in the presence of a certain CO2 level. In some aspects, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 35°C-37°C, about 36°C-37°C, or about 37°C. The CO2 level 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.

[0196] Each step of the method described herein can be performed in a closed system. The closed system can be any suitable cell culture bag (e.g., MACS from Technibio Biotech, Germany). ®GMP cell differentiation bags, Origen Biomedical PermaLife Cell Culture bags) closed bag culture system. The cell culture bags used in the closed bag culture system can be coated with recombinant human fibronectin fragments during the transduction step. The recombinant human fibronectin fragment can include three functional domains: a central cell binding domain, a heparin binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can increase the gene transfer efficiency of retroviral transduction of immune cells by assisting the co-localization of target cells and viral vectors. In one embodiment, the recombinant human fibronectin fragment is RETRONECTIN ® (Takara Bio, Japan). The cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1µg / mL-60µg / mL or about 1µg / mL-40µg / mL, about 1µg / mL-20µg / mL, 20µg / mL-40µg / mL, 40µg / mL-60µg / mL, about 1µg / mL, about 2µg / mL, about 3µg / mL, about 4µg / mL, about 5µg / mL, about 6µg / mL, about 7µg / mL, about 8µg / mL, about 9µg / mL, about 10µg / mL, about 11µg / mL, about 12µg / mL, about 13µg / mL, about 14µg / mL, about 15µg / mL, about 16µg / mL, about 17µg / mL, about 18µg / mL, about 19µg / mL, about 20 g / mL, about 2µg / mL-5µg / mL, about 2µg / mL-10µg / mL, about 2µg / mL-20µg / mL, about 2µg / mL-25µg / mL, about 2µg / mL-30µg / mL, about 2µg / mL-35µg / mL, about 2µg / mL-40µg / mL, about 2µg / mL-50µg / mL, about 2µg / mL-60µg / mL, at least about 2µg / mL, at least about 5µg / mL, at least about 10µg / mL, at least about 15µg / mL, at least about 20µg / mL, at least about 25µg / mL, at least about 30µg / mL, at least about 40µg / mL, at least about 50µg / mL, or at least about 60µg / mL of recombinant human fibronectin fragment. In one embodiment, the cell culture bag is coated with at least about 10 μg / mL recombinant human fibronectin fragment. The cell culture bag used in the closed bag culture system can 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.

[0197] The engineered immune cell population produced by the above method can be optionally cryopreserved so that the cells can be used later. A method for cryopreserving an engineered immune cell population is also provided herein. This method may include the steps of washing and concentrating the engineered immune cell population with a diluent solution. For example, the diluent solution is saline, 0.9% saline, PlasmaLyte A (PL), 5% glucose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In addition, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another aspect, the washing solution is saline, and HSA (5%) is supplemented to the washed and concentrated cells. The method may also include the step of producing a cryopreservation mixture, wherein the cryopreservation mixture includes a diluted cell population in a diluent solution and a suitable cryopreservation solution. The cryopreservation solution may be any suitable cryopreservation solution, including but not limited to CryoStor10 (BioLife Solution), which is mixed with a diluent solution of engineered immune cells at a ratio of 1:1 or 2:1. HSA can be added to provide a final concentration of about 1.0-10%, 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%, about 1%-3% HSA, about 1%-4% HSA, about 1%-5% HSA, about 1%-7% HSA, about 2%-4% HSA, about 2%-5% HSA, about 2%-6% HSA, about 2%-7% HSA, or about 2.5% HSA in the cryopreservation mixture. Cryopreservation of the engineered immune cell population can include washing the cells with 0.9% saline, adding HSA to a final concentration of 5% to the washed cells, and diluting the cells 1:1 with CryoStorTM CS10 (for a final concentration of 2.5% HSA in the final cryopreservation mixture). In some aspects, the method also includes the step of freezing the cryopreservation mixture. Alternatively, the cryopreservation mixture was placed in a controlled rate freezer using defined freezing cycles at a temperature between approximately 1 × 10 6 About 1.5×10 7 The method may further comprise the step of storing the cryopreservation mixture in vapor phase liquid nitrogen.

[0198] The engineered immune cell population produced by the methods described herein can be cryopreserved at a predetermined dose. The predetermined dose can be a therapeutically effective dose, which can be any therapeutically effective dose provided below. The predetermined dose of engineered immune cells can depend on the cell surface receptors expressed by the immune cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cells, the nature of the disease, or the pathological condition being treated, or a combination thereof.

[0199] In one embodiment, the engineered T cell population can be cryopreserved at a predetermined dose of about 1 million engineered T cells / kilogram body weight (cells / kg). In certain embodiments, the engineered T cell population can be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered T cells / kg. In certain embodiments, the engineered T cell population can be cryopreserved at a predetermined dose of at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, at least about 10 million engineered T cells / kg. In other aspects, the engineered T cell group can be cryopreserved with a predetermined dose 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 some aspects, the engineered T cell group can be cryopreserved with a predetermined dose of about 1 million to about 2 million engineered T cells / kg. The engineered T cell group can be cryopreserved with a predetermined dose of about 1 million cells to about 2 million cells / kg, about 1 million cells to about 3 million cells / kg, about 1 million cells to about 4 million cells / kg, about 1 million cells to about 5 million cells / kg, about 1 million cells to about 6 million cells / kg, about 1 million cells to about 7 million cells / kg, about 1 million cells to about 8 million cells / kg, about 1 million cells to about 9 million cells / kg, about 1 million cells to about 10 million cells / kg. The predetermined dose of the engineered T cell group can be calculated based on the body weight of the experimenter. In an example, the engineered T cell group can be cryopreserved in the cryopreservation medium of about 0.5-200mL.In addition, the engineered T cell group can be cryopreserved in the cryopreservation medium of about 0.5mL, about 1.0mL, about 5.0mL, about 10.0mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70mL, about 80mL, about 90mL or about 100mL, about 10mL-30mL, about 10mL-50mL, about 10mL-70mL, about 10mL-90mL, about 50mL-70mL, about 50mL-90mL, about 50mL-110mL, about 50mL-150mL or about 100mL-200mL. In some aspects, the engineered T cell group can be preferably cryopreserved in about 50-70mL cryopreservation medium.

[0200] In one embodiment, serum-free medium without added serum is used to perform at least one of the following: (a) contacting an immune cell population with exogenous IL-2, exogenous IL-7, exogenous IL-15 and / or other cytokines, (b) stimulating an immune cell population, (c) transducing an activated immune cell population, and (d) amplifying a transduced immune cell population. In some aspects, each of (a) to (d) is performed using a serum-free medium without added serum. As mentioned herein, the term "serum-free medium" or "serum-free medium" means 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 culture medium as a separate and distinct component for the purpose of supporting the viability, activation and growth of cultured cells. According to the methods described herein, any suitable immune cell growth medium can be used to culture cells in suspension. For example, immune cell growth medium can include, but is not limited to, a sterile low glucose solution comprising a suitable amount of buffer, magnesium pyruvate, calcium pyruvate, sodium pyruvate and sodium bicarbonate. In one aspect, 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 medium that is not supplemented with serum (eg, human or bovine).

[0201] The present application provides various methods for treating cancer with T cells. In one aspect, the T cells are CAR-T cells for CD19, which can be prepared by any of the methods of the present application in combination with any step of a manufacturing method for preparing T cells for immunotherapy, any of which includes but is not limited to those described in PCT publications WO2015 / 120096 and WO2017 / 070395, which are incorporated herein by reference in their entirety for the purpose of describing these methods; for preparing Akilencel or Yescarta ®Any and all methods for preparing tesalencyclidine / Kymriah ™ Any and all methods for preparing "ready-made" T cells for immunotherapy; Any and all methods for preparing lymphocytes for administration to humans. In some aspects, the manufacturing method is suitable for specifically removing circulating tumor cells from cells obtained from a patient.

[0202] In one aspect, the T cells are CD19 CAR-T cells prepared by the method described in PCT / US2016 / 057983. In one embodiment, a T cell population depleted of circulating tumor cells is prepared from a leukapheresis product. These cells can be prepared as described in 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 subject's T cells are engineered to express a receptor consisting of a single-chain antibody fragment against CD19 linked to CD28 and CD3ζ activation domains, resulting in the elimination of cells expressing CD19. In the CAR and CD19 + Upon target cell engagement, the CD3ζ domain activates downstream signaling cascades that lead to T cell activation, proliferation, and acquisition of effector functions, such as cytotoxicity. The intracellular signaling domain of CD28 provides a co-stimulatory signal that works with the primary CD3ζ signal to enhance T cell function, including interleukin (IL)-2 production. Together, these signals can stimulate proliferation of CAR T cells and directly kill target cells. In addition, activated T cells can secrete cytokines, chemokines, and other molecules that can recruit and activate additional anti-tumor immune cells. The anti-CD19 CAR in the CD19 CAR-T cell can comprise FMC63-28Z.

[0203] Due to the presence of circulating tumor cells in some cancers, the manufacture of CD19 CAR-T cells includes CD4 + and CD8 + T cell enrichment step. The T cell enrichment or isolation step can reduce CD19-expressing circulating tumor cells in the leukapheresis material and can be involved in activating, expanding, and depleting anti-CD19 CAR T cells during manufacturing.

[0204] The methods described herein can enhance the therapeutic outcome or efficacy of immune or cell therapy, which can be adoptive T cell therapy 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 selection, in vitro enrichment and administration of autologous or allogeneic T cells that recognize and can bind to tumor cells to the patient. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes that can infiltrate tumor tissue are separated, enriched in vitro and administered to the patient. TIL cells can be autologous or allogeneic. Autologous cell therapy is adoptive T cell therapy, which involves separating T cells that can target the patient's tumor cells, enriching T cells in vitro and administering T cells back to the same patient. Allogeneic T cell transplantation can include transplantation of naturally occurring T cells or genetically engineered T cells that are amplified ex vivo. Engineered autologous cell therapy as described in more detail above is adoptive T cell therapy in which the patient's own lymphocytes are separated, genetically modified to express tumor targeting molecules, amplified in vitro and administered back to the patient. Non-T cell transplantation can include autologous or allogeneic therapy with non-T cells (such as but not limited to natural killer (NK) cells).

[0205] The immune cell therapy of this application is an engineered autologous cell therapy (eACT ™). According to this aspect, the method may include collecting immune cells from a donor. The isolated immune cells may then be contacted with an exogenous activation agent (e.g., a cytokine), amplified and engineered to express a chimeric antigen receptor ("engineered CAR T cell") or a T cell receptor ("engineered TCR T cell"). In some aspects, the engineered immune cells treat a tumor in a subject. For example, the one or more immune cells are transduced with a retrovirus comprising a heterologous gene 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 (e.g., 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, the 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 an antigen binding molecule thereof. For example, the antigen binding molecule may be selected from scFv, Fab, Fab', Fv, F(ab')2 and dAb and any fragment or combination thereof. The chimeric antigen receptor may also include a hinge region. The hinge region can be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8α. In one embodiment, the hinge region is derived from the hinge region of IgG4. The chimeric antigen receptor can also include a transmembrane domain. The transmembrane domain can be the transmembrane domain of any transmembrane molecule, which is a co-receptor on an immune cell or a transmembrane domain of an immunoglobulin superfamily member. In certain embodiments, the transmembrane domain is derived from the transmembrane domain of the following substances: CD28, CD28T, CD8 α, 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 can also include one or more costimulatory signaling regions. For example, the costimulatory signaling region can be a signaling region of CD28, CD28T, OX-40, 41BB, CD27, inducible T cell co-stimulation (ICOS), CD3γ, CD3δ, CD3ε, CD247, Ig α (CD79a), or Fc γ receptor. In another embodiment, the costimulatory signaling region is a CD28 signaling region. In another embodiment, the costimulatory signaling region is a CD28T signaling region. In an additional embodiment, the chimeric antigen receptor further comprises a CD3ζ signaling domain.

[0206] In some aspects, the tumor antigen is selected from 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (cluster of differentiation region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exon 7 / 8), CAMEL (antigen recognized by CTL on melanoma), CAP-1 (carcinoembryonic antigen peptide 1), CASP-8 (caspase 8), CDC27m (mutated cell division cycle protein 27), CDK4 / m (mutated cyclin-dependent kinase 4), CEA (carcinoembryonic antigen), CT (carcinoma / testis). granules (antigens), Cyp-B (cyclophilin B), DAM (differentiation antigen on melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (mutated elongation factor 2), ETV6-AML1 (Ets mutant somatic gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2 / neu (human epidermal receptor 2 / neural;Also known as EGFR2), HLA-A (human leukocyte antigen A), HPV (human papillomavirus), HSP70-2M (mutated heat shock protein 70-2), HST-2 (human circular body tumor factor 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin 13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), kappa light chain, LAGE (L antigen), LDLR / FUT (low-density lipid receptor / GDP-L-fucose: bD-galactosidase 2-aL fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen recognized by T cells 1 / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (mutated myosin), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitously mutated proteins 1, 2, 3), NA88-A (NA of patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 smaller bcr-abl (190KD protein bcr-abl), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitin 1 or 2), SAGE (sarcoma Antigen), SART-1 or SART-3 (squamous antigen for tumor rejection 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets family leukemia / acute myeloid leukemia 1), TPI / m (mutated triosephosphate isomerase), 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 tumor gene), and any combination thereof. In one embodiment, the tumor antigen is CD19. ;

[0207] T cell therapy involves administering to a patient engineered T cells that express a T cell receptor ("engineered TCRT cells"). The T cell receptor (TCR) may contain a binding molecule to a tumor antigen. In some aspects, the tumor antigen is selected from the group consisting of: 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, KIAA 0205, KDR, kappa light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, mesothelin, murine 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 smaller 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.

[0208] "CD19-directed, genetically modified autologous T-cell immunotherapy" refers to a suspension of chimeric antigen receptor (CAR)-positive immune cells. An example of this type of immunotherapy is Clear CAR-T therapy, which uses CAR-T cells that are free of circulating tumor cells and enriched for CD4+ / CD8+ T cells. Another example is axicabtagene ciloleucel (also known as Axi-cel ™ , 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 (April 2013). To prepare CD19-directed genetically modified autologous T cell immunotherapy, the patient's own T cells can be collected and genetically modified by reverse transcription transduction in vitro to express a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable region fragment (scFv) connected to CD28 and CD3-ζ costimulatory domains. In some embodiments, the CAR contains a mouse anti-CD19 single-chain variable fragment (scFv) connected to 4-1BB and CD3-ζ costimulatory domains. Anti-CD19 CAR T cells can be expanded and infused back into the patient, where they can recognize and eliminate target cells expressing CD19.

[0209] In one aspect, the TCR comprises a binding molecule 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 comprises a binding molecule 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 crack 2 (SSX2), melanoma antigen (MAGE) and any combination thereof. In another embodiment, the TCR comprises a binding molecule to a lineage-specific antigen. In an additional embodiment, the lineage-specific antigen is selected from the group consisting of melanoma antigen 1 (MART-1) recognized by T cells, gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA) and any combination thereof. In certain embodiments, the T cell therapy comprises administering to the patient an engineered CAR T cell expressing a chimeric antigen receptor that binds to CD19 and further comprises a CD28 co-stimulatory domain and a CD3-ζ signaling region. In additional embodiments, the T cell therapy comprises administering to the patient KTE-C19. In one aspect, the antigenic portion further includes, but is not limited to, Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, type 2, or type 8 (HSV1, HSV2, or HSV8) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL 32, US43, UL45, UL49A), cytomegalovirus (CMV) viral antigens (e.g., glycoprotein gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoprotein gp120, gp41 or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) viral antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) virus antigens or varicella-zoster virus antigens. In such aspects, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on a virus-infected target cell. In other aspects, the antigen portion is associated with cells with immune or inflammatory dysfunction.Such antigenic portions may 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 proteins (HSP), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0210] The methods disclosed herein may involve T cell therapy comprising transferring one or more T cells to a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount 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 In another aspect, the therapeutically effective amount of T cells (e.g., 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 In one embodiment, a therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7cells / kg or about 9×10 7 In one embodiment, the amount of CD19 CAR-T cells is 2×10 6 cells / kg, the maximum dose for subjects ≥100 kg is 2×10 8 In another embodiment, the amount of CD19 CAR-T cells is 0.5×10 6 cells / kg, the maximum dose for subjects ≥100 kg is 0.5×10 8 cells.

[0211] The patient may be preconditioned or lymphodepleted before the administration of T cell therapy. The patient may be preconditioned according to any method known in the art, including but not limited to treatment with one or more chemotherapy drugs and / or radiotherapy. In some aspects, preconditioning may include any treatment that reduces the number of endogenous lymphocytes, removes cytokine levels, increases serum levels of one or more homeostatic cytokines or proinflammatory factors, enhances the effector function of T cells administered after conditioning, enhances antigen presenting cell activation and / or availability before T cell therapy, or any combination thereof. Preconditioning may include increasing the serum level of one or more cytokines of the subject. The method also includes administering a chemotherapy drug. The chemotherapy drug may be a lymphodepleting (preconditioning) chemotherapy drug. Beneficial preconditioning treatment regimens are described in U.S. Pat. No. 9,855,298, along with associated beneficial biomarkers, which is hereby incorporated by reference in their entirety. These provisional patent applications describe, for example, methods for regulating patients in need of T cell therapy, the methods comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (200 mg / m 2 / day and 2000mg / m 2 / day) and the specified dose of fludarabine (20 mg / m 2 / day and 900mg / m 2 One such dosage regimen involves treating a patient by administering to the patient about 500 mg / m2 per day for three days prior to administering to the patient a therapeutically effective amount of engineered T cells. 2 / day of cyclophosphamide and about 60 mg / m 2 / day of fludarabine. In one aspect, the conditioning regimen comprises 500 mg / m 2 Cyclophosphamide + 30 mg / m 2 Fludarabine. They can be administered on day -4, day -3 and day -2 or on day -5, day -4 and day -3 (day 0 is the day the cells are administered). In one embodiment, the conditioning regimen includes 200 mg / m 2 , 250mg / m2 , 300mg / m 2 , 400v, 500mg / m 2 Cyclophosphamide for 2, 3, or 4 days and 20 mg / m 2 , 25mg / m 2 or 30 mg / m 2 Fludarabine is continued for 2, 3, or 4 days. In one embodiment, conditioning chemotherapy (30 mg / m per day) is administered after leukapheresis. 2 Fludarabine and 500 mg / m2 / day 2Cyclophosphamide) is administered on days -5, -4, and -3 before intravenous infusion of a suspension of CD19 CAR-T cells. In some embodiments, the intravenous infusion time is between 15 minutes and 120 minutes. In one embodiment, the intravenous infusion time is between 1 minute and 240 minutes. In some embodiments, the intravenous infusion time is at most 30 minutes. In some embodiments, the intravenous infusion time is at most 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes or at most 100 minutes. In some embodiments, the infusion volume is between 50mL and 100mL. In some embodiments, the infusion volume is between 20mL and 100ml. In some embodiments, the infusion volume is about 30mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL or about 65mL. In some embodiments, the infusion volume is about 68mL. In some embodiments, the suspension has been frozen and used within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour of thawing. In some embodiments, the suspension has not been frozen. In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is stirred during the infusion. In some embodiments, the immunotherapy is administered within 3 hours after thawing. In some embodiments, the suspension also includes albumin. In some embodiments, albumin is present in an amount of about 2% to 3% (volume / volume). In some embodiments, albumin is present in an amount of about 2.5% (volume / volume). In some embodiments, albumin is present in an amount of about 1%, 2%, 3%, 4% or 5% (volume / volume). In some embodiments, albumin is human albumin. In some embodiments, the suspension also includes DMSO. In some embodiments, DMSO is present in an amount of about 4% to 6% (vol / vol). In some embodiments, DMSO is present in an amount of about 5% (vol / vol). In some embodiments, DMSO is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (v / v).

[0212] The methods disclosed herein can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent the growth of a tumor, eliminate a patient's tumor, prevent the recurrence of a tumor, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain aspects, the methods can induce a complete response. In other aspects, the methods can induce a partial response.

[0213] Treatable cancers include tumors that are not vascularized, not fully vascularized, or vascularized. Cancers can also include solid tumors or non-solid tumors.

[0214] In one embodiment, the method can be used to treat B-cell malignancies that carry high levels of circulating tumor cells expressing CD19 and would be indicated for a diverse patient population with high unmet need.

[0215] In some embodiments, the CAR T cell intervention comprises T cells that are expanded from a T cell population that is depleted of circulating lymphoma cells and enriched for CD4+ / CD8+ T cells by positively selecting monocytes from a leukapheresis sample that is activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and then transduced with a replication-defective viral vector containing an anti-CD19 CAR construct. In some embodiments, the CAR construct is FMC63-28ZCAR. The CAR T cells produced using this method may be referred to as KTE-X19. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments, the dose of CAR-positive T cells is 2×10 6 Anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR positive T cells is 1×10 6 Anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR positive T cells is 1.6×10 6 Anti-CD19 CAR T cells / kg, 1.8×10 6 Anti-CD19 CAR T cells / kg or 1.9×10 6 In some embodiments, the CD19 CAR construct contains a CD3ζ T cell activation domain and a CD28 signaling domain.

[0216] In some embodiments, after leukapheresis, 25 mg / m2 per day on days -5, -4, and -3 2 Fludarabine and 900 mg / m2 / day on day -2 2 CAR T cells are administered as a single infusion on day 0 following conditioning therapy with cyclophosphamide. In some embodiments, the conditioning therapy comprises 300 mg / m per day for 3 days. 2 Cyclophosphamide and 30 mg / m2 / day 2 Fludarabine. In some embodiments, conditioning chemotherapy includes 30 mg / m2 per day on days -5, -4, and -3. 2 Fludarabine and 500 mg / m2 / day 2Cyclophosphamide. In some embodiments, the patient may also receive acetaminophen and diphenhydramine or another H1 antihistamine approximately 30 to 60 minutes prior to infusion of the anti-CD19 CAR T cells. In some embodiments, the patient receives one or more additional doses of anti-CD19 CAR T cells.

[0217] In some embodiments, the MCL cancer is relapsed / refractory MCL (r / r MCL). In some embodiments, the patient has received one or more previous treatments. In some embodiments, the patient has received 1 to 5 previous treatments. In some embodiments, previous treatments may include autologous SCT, anti-CD20 antibodies, chemotherapy containing anthracyclines or bendamustine, and / or Bruton's tyrosine kinase inhibitors (BTKi). In some embodiments, BTKi is ibrutinib (Ibr). In some embodiments, BTKi is acalabrutinib (Acala). In some embodiments, the present disclosure shows that MCL patients previously treated with ibrutinib have a more significant response to anti-CD19 CAR T cell therapy than patients previously treated with acalabrutinib. Therefore, the present disclosure provides a method for treating r / r MCL with anti-CD19 CAR T cell therapy, wherein the patient has previously been treated with ibrutinib or acalabrutinib and its cancer is preferably relapsed / refractory to ibrutinib or acalabrutinib. In some embodiments, the BTKi is tirabrutinib (ONO-4059), zanubrutinib (BGB-3111), CGI-1746, or spebrutinib (AVL-292, CC-292).

[0218] In some embodiments, the disclosure indicates that for patients who received prior ibrutinib, acalabrutinib, or both, the median (range) peak CAR T cell levels were 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 of anti-CD19 CAR T cell therapy in patients with MCL were 94% / 65% in patients who received prior ibrutinib, 80% / 40% in patients who received prior acalabrutinib, and 100% / 100% in patients who received two BTKis. In some embodiments, the 12-month survival rates in patients who received prior ibrutinib, acalabrutinib, or both were 81%, 80%, or 100%, respectively. In some embodiments, CAR T cell expansion correlates with the ORR / CR rates in patients previously treated with ibrutinib and / or acalabrutinib. Therefore, in one embodiment, patients are treated with both ibrutinib and acalabrutinib. In one embodiment, the present disclosure provides a method for predicting ORR / CR in MCL patients previously treated with ibrutinib and / or acalabrutinib by measuring peak CAR T cell levels and comparing them to reference standards. In one embodiment, the present disclosure provides a method for predicting a progressive response based on measurements of CAR T cell peak levels / baseline tumor burden (CEN and INV). In one embodiment, the higher the ratio, the higher the likelihood of a progressive response at / to 12 months. In one embodiment, a ratio between 0.00001 and 0.005 predicts no response at / to 12 months. In one embodiment, a ratio between 0.006 and 0.3 predicts recurrence at / to 12 months. In one embodiment, a ratio between 0.4 and 1 predicts a progressive response at / to 12 months. In one embodiment, the ratio can be determined by a person of ordinary skill in the art from an average population.

[0219] In some embodiments, the patient may have received bridging therapy (after leukapheresis and before chemotherapy) with dexamethasone (e.g., 20 mg to 40 mg or equivalent given PO or IV daily for 1 to 4 days), methylprednisolone, ibrutinib (e.g., 560 mg given PO daily), and / or acalabrutinib (e.g., 100 mg given PO twice daily) after leukapheresis and, e.g., completed 5 days or less prior to conditioning chemotherapy. In some embodiments, such patients may have a high disease burden. In some embodiments, the bridging therapy is selected from an immunomodulator, R-CHOP, bendamustine, an alkylating agent, and / or a platinum-based agent.

[0220] In some embodiments, the disclosure shows that all MCL patients who responded to CAR T cell infusion achieved T cell expansion, while no expansion was observed in patients who did not respond. In some embodiments, the response is an objective response (complete response + partial response). The disclosure shows that CAR T cell levels correlate with ORR in the first 28 days, where the area under the curve (AUC) from day 0 to day 28 is 0-28 ) and peak levels were >200-fold higher in responders than in non-responders, suggesting that higher expansion leads to better and potentially deeper responses, as also shown by a higher rate of response in minimal residual disease (MRD, 10) compared with MRD-positive patients (at week 4). -5 The peak / AUC CAR T cell level in the negative patient is higher than 80 times as indicated by the sensitivity of the CAR T cell) negative patient. Therefore, the present disclosure provides a method for predicting patient response and MRD to CAR T cell treatment of MCL, the method comprising measuring the peak / AUC CAR T cell level and comparing it to a reference standard. In some embodiments, peak CAR T cell expansion is observed between day 8 and day 15 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. In some instances, the number of CAR T cells is measured in cells / microliter of blood. In some instances, the number of CAR T cells is measured by the number of CAR gene copies / µg of host DNA. In some instances, the number of CAR T cells is measured as described in: Kochenderfer JN et al. J. Clin. Oncol. 2015; 33:540-549. In one embodiment, the level of CAR T cells is measured as described in: Locke FL et al. Mol Ther. 2017; 25(1):285-295.

[0221] In some embodiments, the present disclosure indicates that CAR T cell expansion is higher in patients with grade ≥3 MCL than in patients with grade ≤3 CRS and NE events. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS and NE events, the method comprising measuring CAR T cell expansion after CAR T cell therapy and comparing the level with a reference value, wherein the higher the CAR T cell expansion, the greater the chance of grade ≥3 CRS and NE events.

[0222] In some embodiments, cytokine levels are measured by either protein or mRNA levels. In some embodiments, cytokine levels are measured as described in Locke FL et al. Mol Ther., 2017; 25(1):285-295.

[0223] In some embodiments, the present disclosure shows that serum GM-CSF and IL-6 peak levels (reached about 8 days after CAR T cell administration) are positively correlated with grade ≥3 CRS and grade ≥3 NE in MCL patients. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS and grade ≥3 NE, the method comprising 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 greater the chance of grade ≥3 CRS and NE.

[0224] In some embodiments, the present disclosure shows that serum ferritin is positively correlated with grade ≥3 CRS in MCL patients. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS, the method comprising measuring the peak level of serum ferritin after CAR T cell administration and comparing them with a reference level, wherein the higher the peak ferritin level, the greater the chance of grade ≥3 CRS.

[0225] In some embodiments, the present disclosure indicates that serum IL-2 and IFN-γ are positively correlated with grade ≥3 NE in MCL patients. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS, the method comprising measuring the peak levels of serum IL-2 and IFN-γ after CAR T cell administration and comparing them with reference levels, wherein the higher the peak levels of IL-2 and IFN-γ, the greater the chance of grade ≥3 NE.

[0226] In some embodiments, the present disclosure indicates that cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and vascular cell adhesion molecule (VCAM) are positively correlated with grade ≥3 NE in MCL patients. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS, the method comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecule (VCAM) after CAR T cell administration and comparing them to reference levels, wherein the higher the cerebrospinal fluid level of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecule (VCAM), the greater the chance of grade ≥3 NE.

[0227] In some embodiments, the disclosure indicates that the peak serum levels of cytokines positively correlated with grade ≥3 CRS include IL-15, IL-2 Rα, 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 indicates that the peak serum levels of cytokines associated with grade ≥3 NE include IL-2, IL-1 Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. In some embodiments, the disclosure indicates that cytokines associated with both grade ≥3 CRS and NE include IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B. In some embodiments, the cytokine serum levels peak within 7 days of CAR T cell administration. Therefore, the present disclosure provides a method for predicting grade ≥3 CRS after CAR T cell administration, the method comprising measuring the peak serum levels of IL-15, IL-2 Rα, 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 the levels to a reference standard. Therefore, the present disclosure also provides a method for predicting grade ≥3 CRS and grade ≥3 NE in MCL, the method comprising measuring the peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b and granzyme B after anti-CD19 CAR T therapy and comparing the levels to a reference standard.

[0228] In some embodiments, the disclosure demonstrates a trend toward increased proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1) peak cytokine levels in patients with MCL having mutated TP53 relative to wild-type TP53. Thus, in some embodiments, the disclosure provides a method of improving response to CAR T cell therapy in MCL, the method comprising manipulating the levels of proliferative and / or inflammatory cytokines following CAR T cell administration.

[0229] In some embodiments, the disclosure shows that there is a trend of increased IFN-γ and IL-6 peak levels and increased IL-2 for patients who are MRD-negative at one month after CART cell administration relative to patients who are MRD-positive at one month. Therefore, the disclosure provides a method for predicting whether a patient is MRD-negative in MCL, the method comprising measuring the peak serum level of IFN-γ, IL-6 and / or IL-2 after anti-CD19 CAR T treatment and comparing the level to a reference standard.

[0230] In some embodiments, the present invention relates to a T cell product, wherein the T cells are expanded from a T cell population depleted of circulating lymphoma cells and enriched for CD4+ / CD8+ T cells by positively selecting monocytes from a leukapheresis sample, which is activated with anti-CD3 antibodies and anti-CD28 antibodies in the presence of IL-2 and then transduced with a replication-defective viral vector containing an anti-CD19 CAR construct. In some embodiments, such T cell products can be used to treat ALL, CLL, AML. In some embodiments, the CAR construct is FMC63-28Z CAR. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments, the dose of CAR-positive T cells is 2×10 6 Anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR positive T cells is 1×10 6 Anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR positive T cells is 1.6×10 6 anti-CD19CAR T cells / kg, 1.8×10 6 Anti-CD19 CAR T cells / kg or 1.9×10 6Anti-CD19 CAR T cells / kg. In some embodiments, the CD19 CAR construct contains a CD3ζ T cell activation domain and a CD28 signaling domain. In some embodiments, the T cell product is KTE-X19. In some embodiments, the disclosure indicates that the anti-CAR T cell products prepared as described in the preceding paragraphs can be used in B cell ALL and B cell NHL. In some embodiments, the product characteristics can be selected from the percentage of T cells of a specific subset (naive, central memory, effector and effector memory), the percentage of CD4+ cells, the percentage of CD8+ cells and the CD4 / CD8 ratio. In some embodiments, the product feature is the level of IFNγδ production (pg / mL) in a co-culture of target cancer cells (e.g., Toledo) cells expressing CD19 mixed with anti-CD19 CAR T product cells at a 1:1 ratio. In one embodiment, IFNγ can be measured in the cell culture medium 24 hours after incubation using a qualified ELISA. In some embodiments, one or more of these product characteristics are superior to the product characteristics of anti-CAR T cells prepared by leukapheresis without CD4+ / CD8+ positive cell enrichment. In some embodiments, the superior product characteristics can be selected from an increase in the percentage of cells with a naive phenotype (CD45RA+CCR7+), a decrease in the percentage of cells with a differentiated phenotype (CCR7-), a decrease in the level of IFNγ-producing cells, and an increase in the level of CD8+ cells. In some embodiments, the anti-CD19 T cell product comprises 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 , naive-like T cells (CD45RA+CCR7+). In some embodiments, the product comprises T N In some embodiments, the T cell product is KTE-X19. In some embodiments, KTE-X19 has ≥ 190 pg / mL of IFN-γ production. In certain embodiments, KTE-X19 has ≥ 90% CD3+ cells. In some other embodiments, the percentage of NK cells in KTE-X19 is 0.1% (range 0.0% - 2.8%). In some additional embodiments, the percentage of CD3+ cells in KTE-X19 is 0.1% (range 0.0% - 2.8%). - The percentage of cellular impurities was 0.5% (range 0.3% - 3.9%).

[0231] In some embodiments, the cancer is a relapsed / refractory B-cell ALL. In some embodiments, the patient is ≤21 years old. In some embodiments, the patient is ≤21 years old, weighs ≥10 kg, and has B-cell ALL, which is primary refractory, relapses within 18 months of the first diagnosis, is relapsed / refractory after ≥2 lines of systemic therapy, or is relapsed / refractory after allogeneic stem cell transplantation at least 100 days before registration. In one embodiment, the cancer is a slowly progressing lymphoma or leukemia. In one embodiment, the cancer is an aggressive B-cell lymphoma, which includes diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), mantle cell lymphoma and many types, subtypes and variants of its blastoid variants and B-lymphoblastoid lymphoma. DLBCL can be DLBCL NOS, T cell / histiocyte-enriched large B cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, leg-type EBV-positive DLBCL of the elderly. Other lymphomas of large B cells include primary mediastinal (thymic) LBCL associated with chronic inflammation, DLBCL, lymphomatoid granulomatosis, ALK-positive LBCL, plasmablastic lymphoma, large B cell lymphoma arising in HHV8-associated multicentric Castleman disease, and primary effusion lymphoma. Other types of lymphoma include unclassifiable B-cell lymphoma with features intermediate between DLBCL and Burkitt's lymphoma and unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin's lymphoma, splenic marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma of MALT, nodal marginal zone B-cell lymphoma, hairy cell leukemia, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), Richter's transformation, and primary effusion lymphoma. The cancer can be at any stage from stage 1 to stage 4.

[0232] In some embodiments, the conditioning chemotherapy / lymphodepletion regimen is administered after ≥7 days or 5 half-lives (if shorter) of washout from bridging chemotherapy. In some embodiments, the conditioning chemotherapy / lymphodepletion regimen consists of 25 mg / m 2 given intravenously (IV) daily on days -4, -3, and -2. 2 Fludarabine and 900 mg / m2 IV daily on day -2 2 Cyclophosphamide. On day 0, a single infusion of anti-CD19 CAR T cells may be administered. In some embodiments, additional infusions of anti-CD19 CAR T cells may be administered at a later time. In some embodiments, patients who achieve a complete response to the first infusion may receive a second infusion of anti-CD19 CAR T cells if progression occurs after remission >3 months, provided CD19 expression has been preserved and neutralizing antibodies against the CAR are not suspected.

[0233] In some embodiments, droplet digital polymerase chain reaction can be used to measure the presence, expansion, and persistence of transduced anti-CD19 CAR+ T cells in the blood. In some embodiments, the procedure is performed as described in: Locke FL et al., Mol Ther., 2017; 25(1):285-295. In some embodiments, the disclosure indicates that CAR T cells may not be detectable at the time of relapse. Median peak CAR T cell levels may be measured using 1×10 6 In some embodiments, patients who achieve CR / CRi have a greater median peak expansion than nonresponders, as do patients with undetectable MRD versus detectable MRD. Higher median peak expansion is also observed in patients with ≥ Grade 3 NE compared to patients with ≤ Grade 2 NE. Some patients who relapse may or may not have detectable CD19-positive cells at the time of relapse. In some embodiments, undetectable MRD (defined as <1 leukemic cell / 10,000 viable cells) can be assessed using flow cytometry (NeoGenomics, Fort Myers, FL) according to methods described in: Borowitz MJ, Wood BL, DevidasM, 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.

[0234] In some embodiments, the disclosure shows that the peak IL-15 serum level is lower in patients with ≥3 CRS. In some embodiments, the disclosure shows that the median peak levels of several proinflammatory markers (IFNγIL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, Granzyme B) tend to be higher in patients with ≥3 CRS and patients with ≥3 NE. Therefore, in some embodiments, the disclosure provides a method for predicting whether a patient will have ≥3 CRS by measuring the peak level of serum IL-15 and comparing it with a reference standard. In some embodiments, the disclosure provides a method for predicting whether a patient will have ≥3 CRS and / or ≥3 NE by measuring the peak level of IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1 and / or Granzyme B and comparing it with a reference standard. In some embodiments, the disclosure provides a method for improving anti-CD19 CAR T cell therapy by administering an agent that reduces the level of one or more of these biomarkers.

[0235] Reference levels / standards can be established by any method known to those of ordinary skill in the art. They are used to identify thresholds or groups of values ​​(e.g., quartiles) that can be compared to determine which group the measured value (cytokine level, CAR T cell number, etc.) for each subject falls into or is above or below the threshold. These groups are established by comparisons of selected different populations, as is typical in the art. Depending on where the measured value falls, one can predict many treatment characteristics, such as objective response, CRS grade, NE grade, etc.

[0236] In certain embodiments, the cancer may be selected from a tumor derived from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, cancer, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoma, central nervous system cancer, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, Carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid proliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, embryonal tumor, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma family tumors, extracranial germ cell tumor, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumor, osteosarcoma, gallbladder cancer, stomach (gastric / stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney 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, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract cancer (involving the NUT gene), oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative Myeloid tumors, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma of intermediate differentiation, pineal Somatoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, colorectal cancer, renal cell (kidney) cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric (gastric) cancer, supratentorial primitive neuroectodermal tumors, T-cell lymphoma, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms tumor. In certain embodiments, the cancer is treated with KTE-X19.

[0237] In one embodiment, the method can be used to treat a tumor, wherein the tumor is a lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood are derived from a single type of multipotent hematopoietic stem cell found in the bone marrow. This stem cell produces both bone marrow progenitor cells and lymphocyte progenitor cells, and then the bone marrow progenitor cells and lymphocyte progenitor cells produce various types of white blood cells found in the body. The white blood cells produced by bone marrow progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells and plasma cells. The white blood cells produced by lymphocyte progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes and macrophages. Lymphoma and leukemia may affect one or more of these cell types in patients. In certain embodiments, the tumor is treated with KTE-X19.

[0238] Typically, lymphoma can be divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a heterogeneous cancer group derived from B lymphocytes, T lymphocytes or natural killer cells. In the United States, B cell lymphoma represents 80-85% of reported cases. In 2013, it was estimated that approximately 69,740 new NHL cases associated with the disease and more than 19,000 deaths occurred. Non-Hodgkin's lymphoma is the most common hematological malignancy and the seventh leading site of new cancers in men and women, and accounts for 4% of all new cancer cases and 3% of cancer-related deaths. In certain embodiments, the lymphoma is treated with KTE-X19.

[0239] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for about 30% of NHL cases. There are about 22,000 newly diagnosed DLBCL in the United States each year. It is classified as an aggressive lymphoma, most of which are cured with conventional chemotherapy (NCCN Guidelines NHL 2014). The first-line therapy for DLBCL usually includes an anthracycline-containing regimen with rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), which has an objective response rate of about 80% and a complete response rate of about 50%, wherein about one-third of patients suffer from refractory disease to initial therapy or relapse after R-CHOP. For those patients who relapse after responding to the first-line therapy, about 40%-60% of patients can achieve a second response with additional chemotherapy. The standard of care for the second-line therapy of eligible patients 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), each of which has an objective response rate of about 63% and a complete response rate of about 26%. Patients who respond to the second-line therapy and are considered to be suitable for transplantation receive a combination of high-dose chemotherapy and ASCT, which is cured in about half of the transplanted patients. Patients who fail ASCT have a very poor prognosis and no cure option. Compared with DLBCL, primary mediastinal large B-cell lymphoma (PMBCL) has different clinical, pathological and molecular characteristics. PMBCL is believed to be produced by thymic (medullary) B cells and represents about 3% of patients diagnosed with DLBCL. PMBCL is usually identified in the 40th decade of life in younger adult populations, with women slightly dominating. Gene expression profiles indicate that PMBCL overlaps with Hodgkin lymphoma in deregulated pathways. Initial therapy for PMBCL typically includes an anthracycline-containing regimen with rituximab, such as infusion-dose adjustable etoposide, doxorubicin, and cyclophosphamide and vincristine, prednisone, and rituximab (DA-EPOCH-R), with or without radiation therapy to the affected area. Follicular lymphoma (FL), a B-cell lymphoma, is the most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHLs. Some patients with FL will histologically transform (TFL) to DLBCL, which is more aggressive and associated with poor outcomes. Histological transformation to DLBCL occurs at an annual rate of approximately 3% for 15 years, with the risk of transformation continuing to decline in subsequent years. The biological mechanism of histological transformation is unknown. Initial treatment of TFL is influenced by previous therapies for follicular lymphoma but typically includes an anthracycline-containing regimen with rituximab to eliminate the aggressive component of the disease.The treatment options for relapsed / refractory PMBCL and TFL are similar to those in DLBCL. In view of the low prevalence of these diseases, a large number of prospective randomized studies have not been conducted in these patient populations. Patients with chemotherapy-refractory diseases have similar or worse prognosis than patients with refractory DLBCL. For example, subjects with refractory, aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have major unmet medical needs, and further research is needed with novel treatments in these populations. In certain embodiments, the DLBCL is treated with KTE-X19.

[0240] The CAR T cell therapy of the present disclosure can be administered as a first line of treatment or a second or later line of treatment. In some embodiments, the CAR T cell therapy is administered as a third line, a fourth line, a fifth line, and the like. The previous line of therapy can be any previous anticancer therapy, including but not limited to Bruton's tyrosine kinase inhibitor (BTKi), checkpoint inhibitors (e.g., anti-PD1 antibodies, pembrolizumab (Keytruda), cemiplizumab (Libtayo), nivolumab (Opdivo); anti-PD-L1 antibodies, atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi); anti-CTLA-4 antibodies, ipilimumab (Yervoy)), anti-CD19 antibodies (e.g., blinatumomab), anti-CD52 antibodies (e.g., alemtuzumab); allogeneic stem cell transplantation, anti-CD20 antibodies (e.g., rituximab), systemic chemotherapy, rituximab, anthracyclines, ofatumumab, and combinations thereof. Previous therapy can also be used in combination with the CD19 CAR T therapy of the present application. In one aspect, eligible patients may suffer from a disease that is refractory to the most recent therapy or relapse within 1 year after autologous hematopoietic stem cell transplantation (HSCT / ASCT). CAR T cell therapy can be administered to patients with or suspected of having refractory and / or relapsed cancer to one or more lines of previous therapy. Cancer may be refractory to the first line of therapy (i.e., primary refractory) or refractory to one or more lines of therapy. , Cancer may relapse twelve months after the first remission, relapse or be refractory after two or more lines of previous therapy, or relapse after HSCT / ASCT. In some embodiments, cancer is refractory to ibrutinib or acalabrutinib. In some embodiments, cancer is NHL, and the disease must be primary refractory, relapsed / refractory after two or more lines of systemic therapy, or relapsed / refractory after autologous or allogeneic stem cell transplantation for ≥100 days and ≥4 weeks before enrolling in CAR T cell therapy. In certain embodiments, the CAR T cell therapy is KTE-X19.

[0241] Thus, the method can be used to treat lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. 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 (monocytoid B-cell lymphoma), splenic diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt's lymphoma, and lymphoblastic lymphoma. In some aspects, the lymphoma or leukemia is selected from B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (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 follicle-centered lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus-positive DLBCL, lymphocytic granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8-related multicentric Large B-cell lymphoma arising in Castleman disease, Burkitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, 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 aspects, the cancer is refractory to one or more previous treatments, and / or the cancer has relapsed after one or more previous treatments. In certain embodiments, KTE-X19 is used to treat leukemia or lymphoma.

[0242] 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 another embodiment, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is refractory to one or more of 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 has relapsed after the one or more therapies. In one embodiment, the cancer is refractory diffuse large B-cell lymphoma. In certain embodiments, the cancer is treated with KTE-X19.

[0243] 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 not otherwise specified (DLBCL NOS), primary mediastinal large B-cell lymphoma, Burkitt's lymphoma (BL), Burkitt's lymphoma, or unclassified B-cell lymphoma between DLBCL and BL. In some embodiments, the cancer is relapsed / refractory. In some embodiments, KTE-X19 treatment is administered as a first-line, second-line therapy, or after 1 or more previous lines of therapy. In some embodiments, the patient is a pediatric patient, an adolescent patient, an adult patient, a patient less than 65 years old, over 65 years old, or any other age group.

[0244] In some embodiments, the compositions comprising immune cells disclosed herein can be administered in combination with any number of additional therapeutic agents. In one embodiment, the additional therapeutic agent is administered simultaneously with the T cell therapy. In one embodiment, the additional therapeutic agent is administered before, during, and / or after the T cell therapy. In one embodiment, one or more additional therapeutic agents are administered prophylactically. In one aspect, the compositions comprising immune cells are administered in combination with agents for the management of adverse events (many of which are described elsewhere in this application, including the Examples section). These agents can manage one or more signs and symptoms of adverse reactions such as fever, hypotension, tachycardia, hypoxia, and chills, including arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, vertigo, aphasia, delirium, insomnia anxiety, allergic reactions, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0245] Examples of such agents include, but are not limited to, tocilizumab, steroids (e.g., methylprednisolone), rabbit antithymocyte globulin. In some aspects, vancomycin and aztreonam (1 mg each IV twice daily) may be administered for non-neutropenic fever. In some aspects, the method further comprises administering a non-sedating anti-epileptic drug for epilepsy prevention; administering at least one of erythropoietin, darbepoetin alfa, platelet transfusion, filgrastim or pegfilgrastim; and / or administering tocilizumab, siltuximab. In one aspect, the agent 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 a variety of hematopoietic and non-hematopoietic cell types when stimulated, and it can activate / "start" myeloid cell populations to produce inflammatory mediators such as TNF and interleukin 1β (IL1β). In some embodiments, a GM-CSF inhibitor is an antibody that binds to and neutralizes circulating GM-CSF. In some embodiments, the antibody is selected from lenzilumab; namezumab (AMG203); GSK3196165 / MOR103 / otilizumab (GSK / MorphoSys), KB002 and KB003 (KaloBios), MT203 (Micromet and Nycomed), and MORAb-022 / jinsilumab (Morphotek). In some embodiments, the antibody is a biosimilar of the antibody. 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-limiting 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 / Latuzumab), or a biosimilar version of any of said antibodies. In some embodiments, the inhibitor or antagonist inactivates the activity of the GM-CSF-R-α (also known as CSF2R) or CSF1R receptor.In some embodiments, the inhibitor is selected from Mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor alpha monoclonal antibody currently being developed by MedImmune; Cabiratumumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly), Imituzumab, also known as 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), 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 series, pyridopyrimidine series); BLZ945 (Novartis), PLX7486, ARRY-382, Pexidrtinib (also known as PLX3397), or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine; GW 2580 (CAS 870483-87-7), ΚΪ20227 (CAS 623142-96-1), AC708 provided by Ambit Siosciences, or any CSF1R inhibitor listed in Cannarile et al., Journal for Immunotherapy of Cancer. of Cancer) 2017, 5:53 and US20180371093, which are incorporated herein by reference for the purpose of their disclosure of inhibitors.Additional neutralizing antibodies to GM-CSF or its receptor have been described in the art, including, for example, "GM-CSF as a target ininflammatory / 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, AW, Nat. Rev. Rheumatol., 2016. In other embodiments, the agent is an anti-IL6 or anti-IL6 receptor blocker, including tocilizumab and siltuximab.

[0246] In one aspect, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzotepa, carboquinone, metodepa, and uredepa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. resume); nitrogen mustards such as chlorambucil, naphthyl mustard, cholephosphamide, estramustine, ifosfamide, dichloromethyl diethylamine, methoxychlor hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosfamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calicheamicin, karubicin, Carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, detoxibicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, noramycin, olivemycin, peplomycin, porfiromycin, puromycin, triferon-adriamycin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zoloft, daunorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5- FU); folic acid analogs such as dimethoate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgen such as caprotestosterone, drostanolone propionate, cyclothiocarb, melastane, testolactone; antiadrenergic such as aminoglutethimide, mitotane, trilosin stan; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; amsacrine; busamustine; bisantrene; edatrexate; defosfamide; colcemid; diacrylamide; elformithine; elformithine; etogluconate; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; diamine nitracrine; pentostatin; methambucil; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK ®; razoxane; sizoran; spirogermanium; tricholomanic acid; triazoline quinone; 2, 2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOLTM, Bristol-Myers Squibb) and docetaxel (TAXOTERE ® , Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; nosocomial; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM (alitretin); ONTAKTM (denileukin); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some aspects, the composition comprising the immune effector cells expressing CAR and / or TCR disclosed herein can be co-administered with an anti-hormonal agent for regulating or inhibiting the hormonal effects on tumors, such as an anti-estrogen, including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles that inhibit aromatase, 4-hydroxytamoxifen, troxifene, raloxifene hydrochloride, LY117018, onastarstone and toremifene (Faloteton); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. Where appropriate, a combination of chemotherapeutic agents is also administered, including but not limited to CHOP, i.e., cyclophosphamide (Cytoxan ® ), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin ® ) and prednisone.

[0247] These chemotherapeutic agents can be used while or within one week after the engineered cell or nucleic acid is used. In other aspects, the chemotherapeutic agent is used 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 engineered cell or nucleic acid is used. In some aspects, the chemotherapeutic agent is used at least 1 month before the cell or nucleic acid is used. In some aspects, the method also includes the use of two or more chemotherapeutic agents.

[0248] A variety of additional therapeutic agents may be used in conjunction with the compositions or agents / 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 GM-CSF, CSF1, GM-CSFR, or CSF1R (anti-CSF1 antibodies selected from those manufactured by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110 / lacnotuzumab)), mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor α monoclonal antibody recently developed by MedImmune, Inc.; cabilutuzumab (Five Primers, Inc.), and GM-CSF receptor α monoclonal antibody (GM-CSF1R). Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly), imipenem, also known as 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 aspects, the inhibitor or antagonist is expressed in CAR-T cells. In some aspects, the inhibitor is a small molecule (e.g., heteroarylamide, quinolinone series, pyridopyrimidine series); BLZ945 (Novartis), PLX7486, ARRY-382, Pexidrtinib (also known as PLX3397) or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine; GW 2580 (CAS 870483-87-7), ΚΪ20227 (CAS 623142-96-1), AC708 provided by Ambit Siosciences, or any CSF1R inhibitor listed in the following literature: Cannarile et al., Journal for Cancer Immunotherapy (Journal for ImmunoTherapy of Cancer, 2017, 5:53 and US20180371093, which are incorporated herein by reference for the purpose of their disclosure of inhibitors. Additional neutralizing antibodies against GM-CSF or its receptors have been described in the art.Additional therapeutic agents suitable for use in combination with the compositions or medicaments / treatments and methods disclosed herein include, but are not limited to, Ibrutinib (IMBRUVICA). ® ), ofatumumab (ARZERRA ® ), rituximab (RITUXAN ® ), bevacizumab (AVASTIN ® ), HERCEPTIN ® ), trastuzumab emtansine (KADCYLA ® ), imatinib (GLEEVEC ® ), Cetuximab (ERBITUX ® ), panitumumab (VECTIBIX ® ), catumaxomab, ibritumomab tiuxetan, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, lenalidomide, axitinib, masitinib, pazopanib, sunitinib, sorafenib, tocilizumab, toranib, lestaurine, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semasanib, sorafenib, sunitinib, tivozanib, toranib, vandetanib, en tretinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestautinib, ruxolitinib, paclitinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegi and vimodegi, CDK inhibitors such as CDK inhibitors (palbociclib).

[0249] Compositions or agents / treatments comprising immune cells may be administered with or may be administered with anti-inflammatory agents. Anti-inflammatory agents or anti-inflammatory drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, corticosteroids, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL ® ), adalimumab (HUMIRA ® ) and infliximab (REMICADE ® ), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecule. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auranofin) and intramuscular) and minocycline.

[0250] The compositions or agents / treatments described herein may be administered in combination with cytokines and / or cytokine modulators as additional therapeutic agents. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO, Epogen ® 、Procrit ®); osteoinductive factors; interferons, such as interferons α, β and γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, 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 factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines. In one embodiment, the compositions described herein are administered in combination with a steroid or a corticosteroid.

[0251] Corticosteroid treatment can be used to treat adverse events. Before any symptoms of adverse events are detected and / or after adverse events are detected, corticosteroids (or any other steroids and any other treatment of adverse events) can be used prophylactically. They can be used before T cells are administered, on the day of T cell administration (before, after and / or during T cell administration) and / or after T cell administration for one or more days. They can be used before, during or after conditioning therapy. Any corticosteroid can be appropriate for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, these two substances are administered in combination. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids.Exemplary glucocorticoids include, but are not limited to, alclomethasone, algestrone, 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), clobetasone, clocortolone (e.g., clocortolone pivalate), cloprednisolone, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortivazole, deflazacort, desonide, desoximetasone, dexamethasone (e.g., dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodium phosphate), diflorasone (e.g., diflorasone diacetate), diflucortolone, difluprednate, glycyrrhetinic acid, fluzacort, fluclosan, fludrocortisone (e.g., fludrocortisone acetate), flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetate), fluocinolone acetate, fluocortidine, fluocortolone, fluorometholone (e.g., fluorometholone acetate), fluperone (e.g., fluperone acetate), fluprednisone, fluprednisolone, fluticasone (e.g., fluticasone propionate), formocort, halcinonide, halobetasol, halometasone, halopediol, hydrocortisone Cortamater, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone acepromate, hydrocortisone acetate, hydrocortisone propylbutyrate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone propylbutyrate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate, maprednione, medrysone, methylprednisone, methylprednisolone (methylprednisolone acepromate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), paramethasone (e.g., paramethasone acetate ), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesate, prednisolone hemisuccinate, prednisolone-21 (β-D-glucuronide), prednisolone meta-sulfobenzoate, prednisolone stiriped, prednisolone butyrate, prednisolone tetrahydrophthalate), prednisone, prednisolone valerate, prednilide, rimexolone, tixocortolone, triamcinolone (e.g., triamcinolone acetonide, triamcinolone acetonide benzoate, triamcinolone hexyl, triamcinolone acetonide 21 palmitate, triamcinolone diacetate).These glucocorticoids and their salts are discussed in detail in, for example, the following documents: Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (1980, 16th edition) and Remington: The Science and Practice of Pharmacy, 22nd edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) and any other editions, which are hereby incorporated by reference. In some embodiments, the glucocorticoid is selected from cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a mineralocorticoid. Any other steroid can be used in the methods provided herein.

[0252] The one or more corticosteroids may be administered at any dosage and frequency of administration that can be adapted to the severity / grade of the adverse event (e.g., CRS and NE). In another embodiment, corticosteroid administration comprises oral or IV administration of 10 mg dexamethasone 1 to 4 times per day. Another embodiment (sometimes referred to as "high-dose" corticosteroids) comprises IV administration of 1 g methylprednisone per day alone or in combination with dexamethasone. In some embodiments, the one or more corticosteroids are administered at a dose of 1 to 2 mg / kg per day.

[0253] Corticosteroids may be administered in any amount effective to improve one or more symptoms associated with an adverse event, such as with CRS or neurotoxicity. Corticosteroids (e.g., glucocorticoids) can be administered, for example, to a 70 kg adult subject in an amount of between or about between 0.1 and 100 mg, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to 40 mg, 0.1 to 30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg, 0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg, or 1 to 10 mg per dose. Typically, a corticosteroid (such as a glucocorticoid) is administered to an average adult subject in an amount between or about between 0.4 and 20 mg, for example, at or about 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 per dose.

[0254] In some embodiments, the dosage may be, for example, at or about 0.001 mg / kg (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, 0.08 mg / kg, 0.085 mg / kg, 0.09 mg / kg, 0.0 Corticosteroids are administered to an average adult subject, typically weighing about 70 to 75 kg, at a dosage of 95 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.50 mg / kg, 0.55 mg / kg, 0.60 mg / kg, 0.65 mg / 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.

[0255] In general, the dose of corticosteroid administered depends on the specific corticosteroid, as there are differences in potency between different corticosteroids. It is generally understood that the potency of drugs varies, so to obtain an equivalent effect, the dose will vary. The equivalence of the potency of various glucocorticoids and routes of administration is well known. Information related to equivalent steroid administration (in a non-chronotherapy manner) can be found in the British National Formulary (BNF) 37, March 1999.

[0256] In some embodiments, the adverse event / reaction may be selected from one or more of the following:

[0257]

[0259] Other adverse reactions include: gastrointestinal disorders: dry mouth; infections and infestations: 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; vascular disorders: bleeding.

[0260] In one embodiment, symptoms of cytokine release syndrome include, but are not limited to, fever, chills, fatigue, anorexia, myalgia, arthalgia, nausea, vomiting, headache, rash, diarrhea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, tachycardia, hypotension, wide pulse pressure, early increase in cardiac output, late decrease in cardiac output, hallucinations, tremors, gait changes, seizures, and death. In one embodiment, methods for grading CRS are 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, neurotoxicity / neurological events can be graded by the methods described in Lee et al., Blood 2014; 124:188-195.

[0261] In some embodiments, adverse events are managed with tocilizumab (or another anti-IL6 / IL6R agent / antagonist), corticosteroid therapy, or anti-epileptic drugs for toxicity prevention. In some embodiments, adverse events are managed by one or more agents selected from inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, anti-thymocyte globulin, lenzilumab, mavrilimumab, cytokines, and anti-inflammatory agents.

[0262] In some embodiments, the disclosure provides methods for preventing the development of adverse reactions to T cell therapy of the disclosure or reducing its severity. In some embodiments, cell therapy is administered together with one or more agents for preventing, delaying the onset of adverse events, reducing the symptoms of adverse events, and treating adverse events, so that the adverse events include cytokine release syndrome and neurotoxicity. In one embodiment, the agent has been described above. In other embodiments, the agent is described below. In some embodiments, the agent is administered before, after, or simultaneously with the administration of cells by one of the methods and dosages described elsewhere in this specification. In one embodiment, the agent is administered to a subject who may be susceptible to a disease but has not yet been diagnosed with the disease.

[0263] In this regard, the disclosed methods may include administering a "prophylactically effective amount" of tocilizumab, corticosteroid therapy, and / or anti-epileptic drugs for toxicity prevention. In some embodiments, the method includes administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, renzilumab, maverickumab, cytokines, and / or anti-inflammatory agents. The pharmacological and / or physiological effect may be preventive, i.e., the effect completely or partially prevents the disease or its symptoms. A "prophylactically effective amount" may refer to an amount that is effective (in dosage and for a necessary period of time) to achieve a desired preventive outcome (e.g., preventing the onset of an adverse reaction).

[0264] In some embodiments, the method includes the management of adverse reactions in any subject. In some embodiments, adverse reactions are selected from cytokine release syndrome (CRS), neurotoxicity, hypersensitivity, severe infection, cytopenia, and hypogammaglobulinemia. In some embodiments, the signs and symptoms of adverse reactions are selected from fever, hypotension, tachycardia, hypoxia, and chills, including arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), epilepsy, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, allergic reaction, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are identified and selected based on one or more biomarkers in the biomarkers of adverse events.

[0265] In some embodiments, the method includes preventing or reducing the severity of CRS in chimeric receptor therapy. 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 manifestations. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. Patients with grade ≥2 CRS (e.g., hypotension, hypoxia that does not respond to fluid resuscitation, or hypoxia requiring oxygen supplementation) should be monitored using continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients with severe CRS, consider performing an echocardiogram to assess cardiac function. For severe or life-threatening CRS, intensive care support therapy may be considered. In some embodiments, the method includes monitoring the patient's CRS signs and symptoms at least daily for 7 days at a certified medical institution after infusion. In some embodiments, the method includes monitoring the patient's signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method includes advising the patient to seek medical attention immediately if signs or symptoms of CRS occur at any time. In some embodiments, at the first sign of CRS, treatment is with supportive care, tocilizumab, or tocilizumab and corticosteroids.

[0266] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients with grade ≥2 neurotoxicity should be monitored using continuous cardiac telemetry and pulse oximetry. Intensive care supportive therapy is provided for severe or life-threatening neurotoxicity. In some embodiments, the symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

[0267] In some embodiments, the cell therapy is administered before, during / simultaneously with, and / or after the administration of one or more agents (e.g., steroids) or treatments (e.g., plaque removal) that treat and / or prevent (preventive) one or more symptoms of adverse events. A preventive effective amount refers to an amount that is effective at a dose and within a necessary time period to achieve the desired preventive result. In one embodiment, a preventive effective amount is used for a subject before or at an earlier stage of the disease. In one embodiment, the preventive effective amount will be less than the therapeutic effective amount. In one embodiment, adverse event treatment or prevention is administered to any patient who will receive, symptomatically receives, or has received cell therapy. In some embodiments, the method of managing adverse events includes monitoring the patient's signs and symptoms of neurotoxicity at least daily for 7 days at a certified medical facility after infusion. In some embodiments, the method includes monitoring the patient's signs or symptoms of neurotoxicity and / or CRS for 4 weeks after infusion.

[0268] In some embodiments, the present disclosure provides two methods for managing adverse events in subjects treated with CAR T cells of steroids and anti-IL6 / anti-IL-6R antibodies. In one embodiment, the present disclosure provides a method for managing adverse events, wherein if there is no improvement after 3 days and for all ≥1 grade neurological events, corticosteroid therapy is initiated for the management of all cases of grade 1 CRS. In one embodiment, if there is no improvement after 3 days and for all ≥2 grade neurological events, tocilizumab is initiated for all cases of grade 1 CRS. In one embodiment, the present disclosure provides a method for reducing overall steroid exposure in patients receiving adverse event management after CAR T cell administration, the method including if there is no improvement after 3 days and for all ≥1 grade neurological events, corticosteroid therapy is initiated for the management of all cases of grade 1 CRS, and / or if there is no improvement after 3 days and for all ≥2 grade neurological events, tocilizumab is initiated for all cases of grade 1 CRS. In one embodiment, corticosteroids and tocilizumab are administered with a regimen selected from those illustrated in the Examples section. In one embodiment, the present disclosure demonstrates that earlier steroid use is not associated with an increased risk of serious infection, reduced CAR T cell expansion, or reduced tumor response.

[0269] In one embodiment, the present disclosure supports the safety of levetiracetam prevention in CAR T cell cancer treatment. In one embodiment, the cancer is NHL. In one embodiment, the cancer is R / R LBCL, and the patient receives KTE-X19. Therefore, in one embodiment, the present disclosure provides a method for managing adverse events in patients treated with CAR T cells, the method comprising administering a prophylactic dose of an anti-epileptic drug to the patient. In some embodiments, if a neurological event occurs after interrupting prophylactic levetiracetam, the patient starts on day 0 of CAR T cell treatment (after conditioning) and also receives levetiracetam (e.g., 750 mg orally or intravenously twice a day) at the onset of ≥ grade 2 neurotoxicity. In one embodiment, if the patient does not experience any ≥ grade 2 neurotoxicity, levetiracetam is gradually reduced and interrupted as clinically indicated. In one embodiment, levetiracetam prevention is combined with any other adverse event management regimen.

[0270] In one embodiment, patients may begin receiving levetiracetam (750 mg orally or intravenously twice daily) on day 0. At the onset of a grade ≥2 neurologic event, the levetiracetam dose is increased to 1000 mg twice daily. In one embodiment, if the patient does not experience any grade ≥2 neurologic events, levetiracetam is tapered and discontinued as clinically indicated. Patients also receive tocilizumab (8 mg / kg [not to exceed 800 mg] given IV over 1 hour) on day 2. Further tocilizumab (± corticosteroids) may be recommended at the onset of grade 2 CRS in patients with comorbidities or elderly age or in the setting of grade ≥3 CRS. Tocilizumab is initiated for patients who experience grade ≥2 neurologic events, and corticosteroids are added for patients with comorbidities or elderly age, or if there is any occurrence of grade ≥3 neurologic events if symptoms worsen despite tocilizumab.

[0271] In one embodiment, the present disclosure shows that prophylactic steroid use appears to reduce the rate of severe CRS and NE to a similar degree as early steroid use administration. Therefore, the present disclosure provides a method for managing adverse events in CAR T cell therapy, wherein patients receive PO dexamethasone 10 mg on day 0 (before infusion), day 1, and day 2. Steroids can also be administered starting with grade 1 NE and grade 1 CRS when no improvement is observed after 3 days of supportive care. If no improvement is observed after 24 hours of supportive care, tocilizumab can also be administered for the management of ≥ grade 1 CRS. In one embodiment, the present disclosure shows that adverse event management of CAR T cell therapy with antibodies that neutralize and / or deplete GM-CSF prevents or reduces treatment-related CRS and / or NE in treated patients. In one embodiment, the antibody is lenzilumab.

[0272] In some embodiments, adverse events are managed by administering one or more agents that are antagonists or inhibitors of IL-6 or IL-6 receptor (IL-6R). In some embodiments, the agent 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 agent is or includes tocilizumab (atlizumab) or salimumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Patent 8,562,991. In some cases, the agent targeting IL-6 is an anti-TL-6 antibody, such as siltuximab, escitumomab, ALD518 / BMS-945429, silukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101 or olozumab (CDP6038) and combinations thereof. In some embodiments, the agent can neutralize IL-6 activity by inhibiting ligand-receptor interactions. In some embodiments, the IL-6 / IL-6R antagonist or inhibitor is an IL-6 mutant protein, such as the IL-6 mutant protein described in U.S. Patent 5,591,827. 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.

[0273] In some embodiments, other agents useful for managing adverse reactions and their symptoms include antagonists or inhibitors of cytokine receptors or cytokines. 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 such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-β receptor (TGF-β I, II or III), IFN-γ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF α 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 agent includes siltuximab, salimumab, olotuzumab (CDP6038), icimumab, ALD518 / BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the agent is an antagonist or inhibitor of a cytokine, such as transforming growth factor β (TGF-β), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNF α, IL-1, interferon γ (IFN-γ), or monocyte chemoattractant protein-I (MCP-1). In some embodiments, the agent is an agent that targets a cytokine receptor (e.g., inhibits a cytokine receptor or acts as an antagonist of a cytokine receptor), such as a TL-6 receptor (IL-6R), an IL-2 receptor (IL-2R / CD25), a MCP-1 (CCL2) receptor (CCR2 or CCR4), a TGF-β receptor (TGF-β I, II or III), an IFN-γ receptor (IFNGR), a MIP1P receptor (e.g., CCR5), a TNF α receptor (e.g., TNFR1), an IL-1 receptor (IL1-Ra / IL-1RP) or an IL-10 receptor (IL-10R), and combinations thereof. In some embodiments, the agent is administered before, after, or simultaneously with the administration of the cells by one of the methods and doses described elsewhere in this specification.

[0274] In some embodiments, the agent is administered at a dose of about 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg, 2 mg / kg to 4 mg / kg, or 6 mg / kg to 8 mg / kg (all inclusive), or at a dose of at least or about 2 mg / kg, 4 mg / kg, 6 mg / kg, or 8 mg / kg. In some embodiments, the agent is administered at a dose of about 1 mg / kg to 12 mg / kg (such as or about 10 mg / kg). In some embodiments, the agent is administered by intravenous infusion. In one embodiment, the agent is tocilizumab. In some embodiments, the agent (e.g., specific tocilizumab) is administered before, after, or simultaneously with the administration of cells by one of the methods and doses described elsewhere in this specification.

[0275] In some embodiments, the method includes identifying CRS based on clinical manifestations. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, it can be managed according to the recommendations in Scheme A, which can also be used in combination with other treatments disclosed herein (including neutralization or reduction of the CSF / CSFR1 axis). Patients who develop ≥ grade 2 CRS (e.g., hypotension, hypoxia that is unresponsive to fluid resuscitation, or oxygen supplementation) should be monitored using continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients who develop severe CRS, consider performing an echocardiogram to assess cardiac function. For severe or life-threatening CRS, intensive care supportive therapy may be considered. In some embodiments, in the methods disclosed herein, a biosimilar or equivalent drug of tocilizumab may be used instead of tocilizumab. In other embodiments, another anti-IL6R may be used instead of tocilizumab.

[0276] In some embodiments, adverse events are managed according to the following regimen (Regime A):

[0277]

[0278] (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.

[0279] (b) For management of neurotoxicity, see Plan B.

[0280] (c) For more information, see ACEMTRA ®(Tocilizumab) Prescribing Information, https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). It states that the initial U.S. approval was in 2010.

[0281] Neurotoxicity

[0282] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients with grade ≥2 neurotoxicity should be monitored using continuous cardiac telemetry and pulse oximetry. Provide intensive care supportive therapy for severe or life-threatening neurotoxicity. For any grade ≥2 neurotoxicity, consider non-sedating anti-epileptic drugs (e.g., levetiracetam) for epilepsy prevention. The following treatments may be used in combination with other treatments disclosed herein (including neutralization or reduction of the CSF / CSFR1 axis).

[0283] In some embodiments, adverse events are managed according to the following regimen (Regime B):

[0284]

[0285] Additional safety management strategies with corticosteroids

[0286] Administration of corticosteroids and / or tocilizumab at grade 1 may be considered preventive. Supportive care may be provided in all regimens at all CRS and NE severity levels. In one embodiment of the regimen for the management of adverse events associated with CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only in the case of comorbidities or older age); and / or corticosteroids (only in the case of comorbidities or older age); Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids. In another embodiment of the regimen for the management of adverse events related to 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.

[0287] In one embodiment of the regimen for the management of adverse events associated with NE, tocilizumab and / or corticosteroids are administered as follows: 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 tocilizumab does not improve); Grade 4 NE: tocilizumab; and / or corticosteroids. In another embodiment of the regimen for the management of adverse events associated with NE, tocilizumab and / or corticosteroids are administered as follows: 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 treatment is initiated when CRS grade ≥2 and tocilizumab treatment is initiated when CRS grade ≥2. In one embodiment, corticosteroid treatment is started when CRS grade is ≥1 and tocilizumab treatment is started when CRS grade is ≥1. In one embodiment, corticosteroid treatment is started when NE grade is ≥3 and tocilizumab treatment is started when CRS grade is ≥3. In one embodiment, corticosteroid treatment is started when CRS grade is ≥1 and tocilizumab treatment is started when CRS grade is ≥2. In some embodiments, the prophylactic use of tocilizumab administered on day 2 can reduce the rate of ≥ grade 3 CRS. The one or more corticosteroids can be administered at any dose and frequency of administration that can be adapted to the severity / grade of adverse events (e.g., CRS and NE). In another embodiment, corticosteroid administration includes oral or IV administration of 10 mg dexamethasone, 1 to 4 times a day. Another embodiment (sometimes referred to as "high-dose" corticosteroids) includes IV administration of 1 g of methylprednisone per day alone or in combination with dexamethasone. In some embodiments, the one or more corticosteroids are administered at a dose of 1 to 2 mg / kg per day. Generally speaking, the dose of the corticosteroid administered depends on the specific corticosteroid, because there are differences in efficacy between different corticosteroids. It should be generally understood that the efficacy of drugs varies, so to obtain equivalent effects, the dose will change. The equivalence of the efficacy of various glucocorticoids and routes of administration is well known. Information related to the administration of equivalent steroids (in a non-long-term treatment mode) can be found in the British National Formulary (British National Formulary, BNF) 37 (March 1999). The application also provides the dosage and administration of cells prepared by the method of the present application, such as CD19-directed gene-modified autologous T cell immunotherapy infusion bags, which are included in about 68 mL for infusion of chimeric antigen receptor (CAR) positive T cell suspensions. In some embodiments, CAR T cells for infusion are prepared in about 40 mL.In some embodiments, the CAR T cell product is formulated in a total volume of 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 500 mL, 700 mL, 800 mL, 900 mL, 1000 mL. In one aspect, the dose and administration of cells prepared by the method of the present application (e.g., an infusion bag of CD19-directed genetically modified autologous T cell immunotherapy) contains 1×10 in approximately 40 mL. 6 CAR-T positive cell suspension. The target dose can be between about 1×10 6 With about 2×10 6 The maximum number of CAR-positive live T cells was 2×10 8 CAR-positive live T cells.

[0288] In some embodiments, the dosage form comprises a cell suspension for infusion in a single-use patient-specific infusion bag; the route of administration is intravenous; the entire contents of each single-use patient-specific bag are infused over 30 minutes by gravity or a peristaltic pump. In one embodiment, the dosing regimen is 2.0×10 6 A single infusion of 10 anti-CD19 CAR T cells / kg body weight (±20%), with a maximum dose of 2×10 8 Anti-CD19 CAR T cells (for subjects ≥ 100 kg). In some embodiments, the T cells constituting the dose are CD19 CAR-T cells.

[0289] 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 can 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 prepared by one of the methods of the present application. ™ (YESCARTA ® , Akilenci). CAR T cell amounts, dosing regimens, methods of administration, subjects, cancers that fall within the scope of these methods are described elsewhere in this application, and these methods are administered alone or in combination with another chemotherapeutic agent, with or without preconditioning, to any patient described elsewhere in this application.

[0290] The following examples are intended to illustrate various aspects of the present application. Therefore, the specific aspects discussed should not be interpreted as limiting the scope of the present application. For example, although the following examples are directed to T cells transduced with anti-CD19 chimeric antigen receptors (CARs), those skilled in the art will understand that the methods described herein can be applied to immune cells transduced with any CAR. It is obvious to those skilled in the art that various equivalent changes and modifications can be made without departing from the scope of the present application, and it should be understood that such equivalent aspects will be included herein. In addition, all references cited in this application are hereby incorporated by reference in their entirety, as if fully set forth herein.

[0291] The patents and scientific literature mentioned herein establish the 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 hereby incorporated by reference. All other disclosed references, dictionaries, documents, manuscripts, genome database sequences and scientific literature cited herein are hereby incorporated by reference.

[0292] Other features and advantages of the present disclosure will be apparent from the embodiments.

[0293] Example 1

[0294] This example describes a phase 2 multicenter study (ZUMA-2) evaluating the efficacy of brexucabtagene autoleucel (KTE-X19) in patients with relapsed / refractory mantle cell lymphoma (R / R MCL) who had not been previously treated with a Bruton's tyrosine kinase inhibitor (BTKi).

[0295] This Phase 2 study is a multicenter, open-label study evaluating the efficacy of brexu-cel in patients with R / R MCL. Key eligibility criteria for Cohort 3 (NCT04880434) included patients aged ≥18 years with MCL who had received 1 to 5 prior regimens, including prior chemotherapy containing anthracyclines, bendamustine, or high-dose cytarabine and an anti-CD20 monoclonal antibody, but no prior therapy with a BTKi. A history of prior allogeneic stem cell transplant (alloSCT) was allowed if no donor cells were detected in chimerism at >100 days after alloSCT. Patients received fludarabine 30 mg / m administered on days -5, -4, and -3. 2 / day and cyclophosphamide 500 mg / m 2 / day conditioning chemotherapy followed by a single infusion of 2 × 10 6Bridging therapy with dexamethasone, radiation therapy, specific chemotherapy, or any combination thereof was recommended for all patients in Cohort 3, especially those with rapidly progressive disease, clinical worsening, or high disease burden at screening, at the investigator's discretion.

[0296] The primary endpoint is ORR as assessed by an independent radiology review committee according to the Lugano classification. Secondary endpoints include safety, duration of response, progression-free survival, overall survival, levels of circulating CAR T cells and cytokines, and changes in patient-reported outcomes over time. In the pivotal cohort, EuroQol five-dimensional (EQ-5D) scores will be assessed only until month 6, whereas for cohort 3, EQ-5D and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC-QLQ-C30) scores will be assessed over time. Minimal residual disease in cohort 3 will be assessed until month 24 by next-generation sequencing of ctDNA.

[0297] Approximately 90 patients will be enrolled in Cohort 3, with a target ORR of 75%, assuming the observed ORR will be significantly greater than 57%, a historical control rate based on a systematic literature review and meta-analysis. The primary analysis of Cohort 3 will be conducted after 86 patients have been enrolled and treated with brexu-cel, with the opportunity to assess response 6 months after the first objective response or 9 months after brexu-cel infusion, whichever occurs sooner. ZUMA-2 Cohort 3 is currently enrolling patients at 41 sites in the United States, France, Germany, the Netherlands, Spain, and the United Kingdom.

[0298] Example 2

[0299] This example describes the evaluation of durable responses following brigiorencel (KTE-X19) in relapsed / refractory mantle cell lymphoma (R / R MCL) in the ZUMA-2 study. Figure 1After 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 to 96.7) in all 68 treated patients, a CR rate of 68% (95% CI, 55.2 to 78.5), a median duration of response (DOR) of 28.2 months (95% CI, 13.5 to 47.1), and a median overall survival (OS) of 46.6 months (95% CI, 24.9 to not estimable), which was not reached in patients with CR. To identify factors associated with long-term response to KTE-X19, patient and product characteristics were evaluated by response status 24 months after infusion in ZUMA-2.

[0300] Methods: Key ZUMA-2 eligibility criteria included adults (≥18 years) with R / R MCL who had received 1 to 5 prior regimens, including anthracycline- or bendamustine-containing chemotherapy, anti-CD20 monoclonal antibody, and BTKi, and who underwent leukapheresis and conditioning chemotherapy followed by a single infusion of brexu-cel (2 × 10 6 =Anti-CD19 CAR T cells / kg). The primary endpoint was ORR (objective response rate), which was defined as (complete response (CR) + partial response (PR); assessed by an independent radiology 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 assessment of patient, disease, pharmacokinetics, and product characteristics was reported by response status at 24 months (progressive response vs. relapsed response). Baseline patient and disease characteristics, subsequent therapy, product characteristics, and pharmacological outcomes were assessed by response status at 24 months after brexu-cel infusion: progressive responders: patients with progressive response at their 24-month assessment; relapsed responders: responding patients who relapsed before their 24-month assessment; nonresponders: patients who were nonresponders. DOR was assessed in progressive responders and relapsed responders. Statistical analysis: Time-to-event endpoints were analyzed using the Kaplan Meier method; all subgroup analyses were descriptive.

[0301] Results: At a median follow-up of 35.6 months (range, 25.9-56.3), 74 patients were enrolled and underwent leukapheresis, and 68 received brexu-cel ( Figure 2 ).

[0302] Table 1. Baseline Patient and Disease Characteristics by Response Status at 24 Months

[0303]

[0304] a In ZUMA-2, bridging therapy was received after leukapheresis and before conditioning chemotherapy. A smaller proportion of progressive responders received bridging therapy and had an Eastern Cooperative Oncology Group performance-status score of 1 than did relapsed responders, with the median tumor burden (sum of products of diameters) at baseline being approximately 4 times smaller among progressive responders than among relapsed responders (Table 1). The median number of prior therapies was 3 in both subgroups, with a smaller proportion of progressive responders than among relapsed responders who had received prior platinum therapy (Table 1).

[0305] Table 2. Most Recent Therapy by Response Status at 24 Months

[0306]

[0307] Ibrutinib was more frequently the last prior therapy among progressive responders than among relapsed responders, whereas a similar proportion received acalabrutinib as their last prior therapy (Table 2). The median time from the last prior therapy to brexu-cel infusion was similar among progressive responders and relapsed responders but was more than twice as long among nonresponders, although the small sample size may have contributed to this difference.

[0308] 62 patients achieved CR or partial response; 3 patients did not achieve the 24-month assessment visit and were excluded from this analysis. Of the 59 evaluable patients with a response, 29 (47%) had a progressive response at 24 months (progressive responders), and 30 (48%) relapsed before 24 months (relapsed responders). 6 patients did not respond (non-responders). At baseline, the median age was 65 years, and the median number of prior therapies was 3 in both subgroups. Among progressive responders vs. relapsed responders, 66% vs. 43% had ibrutinib and 14% vs. 13% had acalabrutinib as the last prior therapy, with a median (range) time from the last prior therapy of 63 months (26-748) vs. 64.5 months (22-443). Compared with relapsed responders, smaller proportions of progressive responders received bridging therapy (53% vs. 21%, respectively) and prior platinum therapy (40% vs. 10%); whereas similar proportions received prior bendamustine therapy (53% vs. 45%), prior proteasome inhibitor therapy (37% and 41%), and prior autologous stem cell transplantation (37% vs. 48%).

[0309] At baseline, a greater proportion of progressive responders had an ECOG score of 0 compared with relapsed responders (79% vs. 57%, respectively), and the median (range) tumor burden (SPD) was 935.1 (260-6133) in progressive responders and 4233.6 (386-14390) in relapsed responders. The incidence of high-risk features was similar between progressive and relapsed responders, with 66% and 60% having a baseline Ki-67 proliferation index score of ≥30%, 10% and 10% having a TP53 mutation, 45% and 37% having an elevated lactose dehydrogenase level (≥ULN to ≤1.5 ULN), and 10% and 13% having a high-risk Simplified Mantle Cell Lymphoma International Prognostic Index score (>6), respectively.

[0310] Table 3. Duration of response (DOR) using central readout according to Cheson 2014 (Cohort 1: KTE-X19) (MitT analysis group: subjects with complete response)

[0311]

[0312]

[0313] The median (range) DOR was not reached (46.7-not estimable) in progressive responders with CR (n=28) and was 8.3 months (5-13.6) in relapse responders with CR (n=15, Table 3). The median time to initial response was 1 month (range, 0.9-3.1; n=29) vs. 1 month (range, 0.8-1.7; n=30) for progressive responders vs. relapse responders. The median time to complete response was 3 months (range, 0.9-35.1; n=28) vs. 3 months (range, 0.8-9.0; n=15) for progressive responders vs. relapse responders. The median time to conversion from SD or PR to CR was 2.3 months (range, 1.8-34.1; n=16) vs. 2.4 months (range, 2.0-8.1; n=8) for progressive responders vs. relapse responders.

[0314] The median (95% CI) DOR was 47.1 months (24.8-not estimable) in progressive CR responders with high baseline LDH levels (n=12) and 8.3 months (4.7-NE) in recurrent CR responders with high baseline LDH levels (n=5).

[0315] Table 4. Duration of response (DOR) using central readout according to Cheson 2014 (Cohort 1: KTE-X19) (MitT analysis group: subjects with complete response and high baseline LDH levels)

[0316]

[0317]

[0318] Table 5. Duration of response (DOR) using central readout according to Cheson 2014 (Cohort 1: KTE-X19) (MitT analysis group: subjects with objective response and high baseline LDH levels)

[0319]

[0320]

[0321] Table 6: Subsequent Treatment by Response Status at 24 Months

[0322]

[0323] Among relapsed responders, 67% were receiving subsequent anticancer therapy at the time of data collection, the most common of which were radiation therapy (23%), dexamethasone (23%), rituximab (23%), venetoclax (20%), and lenalidomide (20%; patients may have received multiple prior therapies and multiple lines of subsequent therapy).

[0324]

[0325] The 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 2-fold higher in progressive responders than in relapsed responders, respectively (Table 7). A modest increase in the median [range] total number of infused CCR7+ cells was observed in progressive responders vs. relapsed responders (119.8 [37-249.9] vs. 89.1 [6.1-353.4]), suggesting that the role of continuous memory T-cell differentiation in achieving durable responses needs further investigation.

[0326] Table 8. Summary of Product Characteristics by Response Status at 24 Months

[0327]

[0328] The product characteristics were largely similar in progressive and relapsing responders, with a modest increase in the median total number of infused CCR7+ T cells observed in progressive versus relapsing responders (Table 8).

[0329] Table 9A: Peripheral blood T cell phenotype by responder status at day 7 -CD8+CD27-CD28+, progressive (n= 21), others (n=32)

[0330]

[0331]

[0332] Table 9B: Peripheral blood T cell phenotype by responder status at day 7 -CD8+CCR7-CD45RA+CD27-CD28+, Progressive (n=21), other (n=32)

[0333]

[0334]

[0335] Table 9C: Peripheral blood T cell phenotype by responder status at day 7 -CD4+CD27+CD28-, progressive (n= 21), others (n=32)

[0336]

[0337]

[0338] Table 9D: Peripheral blood T cell phenotype by responder status at day 7 -CD8+CCR7+CD45RA-PD1+, progressive (n=21), others (n=32)

[0339]

[0340]

[0341] Peripheral blood T cells from relapsed and non-responder patients showed a more pronounced CD8+CD27-CD28+ effector memory phenotype compared with patients with progressive responses. Progressive responders were enriched in peripheral CD4 T cells that maintained juvenile CD27+ expression and activated CD8 effector memory T cells.

[0342] After a median follow-up of approximately 3 years, brexu-cel continues to show durable responses, with 47% of responders still in a progressive response 24 months after infusion. Progressive responses were observed in patients with high-risk disease features, suggesting that brexu-cel has the potential to produce durable responses in patients with R / R MCL who generally have a poor prognosis. Ibrutinib was more frequently the last prior therapy in progressive responders versus relapsed responders compared with ibrutinib. In summary, progressive responders had lower ECOG PS scores and lower tumor burdens, as well as less frequent use of prior platinum therapy or bridging therapy and less intense regimens for prior relapses, compared with relapsed responders, suggesting the potential for greater benefit if brexu-cel is given earlier in the course of disease. Median peak and AUC CAR T-cell levels were approximately 2-fold higher in progressive responders than in relapsed responders, suggesting that the extent of CAR T-cell expansion may predict the durability of responses. The modest increase in the median total number of infused CCR7+ cells and maintenance of CD27+ peripheral T cells observed in progressive versus relapsing responders may suggest a potential role for continuous memory T-cell differentiation in achieving durable responses.

[0343] Example 3

[0344] This example describes a Phase 2, open-label, multicenter basket study (ZUMA-25) evaluating the safety and efficacy of brigatinib in adults with rare B-cell malignancies, including Waldenstrom's macroglobulinemia, Reye's transformation, Burkitt's lymphoma, and hairy cell leukemia.

[0345] The primary objective of this study is to evaluate the efficacy of brigiorencel in four rare B-cell malignancies. This study uses a basket study design with separate indication-specific substudies to investigate relapsed / refractory Waldenstrom's macroglobulinemia (r / r WM), relapsed / refractory Richter's transformation (r / r RT), relapsed / refractory Burkitt's lymphoma (r / rBL), and relapsed / refractory hairy cell leukemia (r / r HCL).

[0346] Substudy A: The primary objective of this substudy was to evaluate the efficacy of brigiorencel in participants with r / r WM as determined by the combined rate of complete response (CR) and very good partial response (VGPR) as assessed by central review. Participants received lymphodepleting chemotherapy with fludarabine 30 mg / m^2 / day and cyclophosphamide 500 mg / m^2 / day for 3 consecutive days from day -5 to day -3, followed by 2 rest days (day -2 and day -1), and then 2 × 10^6 anti-CD19 chimeric antigen receptor (CAR) T cells / kg or 1 × 10^6 anti-CD19 CAR T cells / kg or 2 × 10^6 anti-CD19 CAR T cells / kg in subjects >100 kg, respectively. 8 or 1×10 8 A fixed-dose single infusion of brigiorencel with anti-CD19 CAR T cells.

[0347] Substudy B: The primary objective of this substudy was to evaluate the efficacy of brigiorencel for diffuse large B-cell lymphoma-Rick's transformation (DLBCL-RT) in participants with r / r RT as determined by objective response rate (ORR) by central assessment. Participants received lymphodepleting chemotherapy with fludarabine 30 mg / m^2 / day and cyclophosphamide 500 mg / m^2 / day for 3 consecutive days from day -5 to day -3, followed by 2 rest days (day -2 and day -1), and then 2 × 10^6 anti-CD19 CAR T cells / kg or 1 × 10^6 anti-CD19 CAR T cells / kg or 2 × 10^6 anti-CD19 CAR T cells / kg in subjects > 100 kg, respectively. 8 or 1×10 8 A fixed-dose single infusion of brigiorencel with anti-CD19 CAR T cells.

[0348] Substudy C: The primary objective of this substudy was to evaluate the efficacy of brigiorencel in participants with r / r BL as determined by ORR as assessed by central review. Participants received lymphodepleting chemotherapy with fludarabine 30 mg / m^2 / day and cyclophosphamide 500 mg / m^2 / day for 3 consecutive days from day -5 to day -3, followed by 2 rest days (day -2 and day -1), and then 2 × 10^6 anti-CD19 CAR T cells / kg or 1 × 10^6 anti-CD19 CAR T cells / kg or 2 × 10^6 anti-CD19 CAR T cells / kg in subjects > 100 kg, respectively. 8 or 1×10 8 A fixed-dose single infusion of brigiorencel with anti-CD19 CAR T cells.

[0349] Substudy D: The primary objective of this substudy was to evaluate the efficacy of brigiorencel in participants with r / r HCL as determined by ORR as assessed by central review. Participants received 3 consecutive days of fludarabine 30 mg / m^2 / day and cyclophosphamide 500 mg / m^2 / day lymphodepleting chemotherapy from day -5 to day -3, followed by 2 rest days (day -2 and day -1), and then 2 × 10^6 anti-CD19 CAR T cells / kg or 1 × 10^6 anti-CD19 CAR T cells / kg or 2 × 10^6 anti-CD19 CAR T cells / kg in subjects > 100 kg, respectively. 8 or 1×10 8 A fixed-dose single infusion of brigiorencel with anti-CD19 CAR T cells.

[0350] Certain inclusion criteria are common to all indications.1) Male or female 18 years of age or older at the time of signing the informed consent; 2) The presence of toxicity due to previous therapy must be stable and recovered to grade 1 or lower (except for clinically insignificant toxicity, such as alopecia); 3) Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; 4) Adequate blood function (unless lower values ​​can be attributed to underlying diseases), as follows: absolute neutrophil count (ANC) ≥500 / µL, platelet count ≥50,000 / µL, hemoglobin level ≥8g / dL; 5) Absolute lymphocyte count ≥100 / µL; 6) Adequate renal, liver, lung and cardiac function, fixed The definition of hepatic involvement is as follows: 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 liver involvement is demonstrated, total bilirubin level ≤ 1.5 × ULN, except in subjects with Gilbert's syndrome, cardiac ejection fraction ≥ 50% and no evidence of pericardial effusion as determined by echocardiography (ECHO) or multi-gated acquisition scan (MUGA) and no clinically significant electrocardiographic (ECG) findings, no clinically significant 7) The following washout period must be met prior to leukapheresis / enrollment: pharmacological doses (≥5 mg / day of prednisone or equivalent doses of other corticosteroids) of adrenocorticoid therapy must be avoided for 7 days prior to leukapheresis, unless otherwise specified in the subprotocol, BTK inhibitors (e.g., ibrutinib or acalabrutinib) must be avoided for at least 1 week or 5 half-lives (whichever is shorter) prior to leukapheresis, antineoplastic drugs used in previous therapy must be avoided within 1 week or 5 half-lives (whichever is shorter) prior to leukapheresis, Systemic inhibitory / stimulatory immune checkpoint molecule therapies (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists) must be avoided for at least 3 half-lives, alemtuzumab must be avoided for at least 6 months prior to registration, PEG-asparaginase must be avoided for at least 3 weeks prior to registration, cladribine and pentostatin must be avoided for 3 months prior to registration, donor lymphocyte infusion must be avoided within 28 days prior to registration, and any immunosuppressive antibody treatment (e.g., anti-CD20, anti-tumor necrosis factor [TNF], anti-interleukin [IL] 6, or anti-IL6 receptor) must be avoided within 4 weeks prior to registration unless such treatment was included in the previous regimen or bridging regimen, in which case a 7-day washout period is required before leukapheresis; and 8) Female subjects of childbearing potential must have a negative serum or urine pregnancy test (women who have undergone sterilization or have been postmenopausal for at least 2 years are not considered to be of childbearing potential).

[0351] Certain exclusion criteria are common to all indications. 1) Prior CAR therapy or other gene-modified T-cell therapy; 2) Prior treatment with any anti-CD19 therapy; 3) History of severe immediate hypersensitivity reactions to aminoglycosides; 4) History of severe immediate hypersensitivity reactions to cyclophosphamide or fludarabine; 5) Presence or suspicion of fungal, bacterial, viral, or other infections that are uncontrolled or require IV antimicrobial management. Uncomplicated urinary tract infection and uncomplicated bacterial pharyngitis are permitted if they respond to active therapy. Only patients with uncomplicated urinary tract infection and uncomplicated bacterial pharyngitis who meet afebrile criteria (i.e., temperature below 38°C) for at least 24 hours before the investigator confirms the patient's eligibility, are eligible; 6) HIV-positive patients, unless taking appropriate anti-HIV medications, have an undetectable viral load by quantitative polymerase chain reaction (qPCR) and a CD4 count >200 cells / uL; 7) Acute or chronic active hepatitis B or C infection. Subjects with a history of hepatitis infection must have cleared their infection as determined by standard serological and genetic testing according to current Infectious Diseases Society of America guidelines or applicable national guidelines; 8) Any indwelling line or drainage tube (e.g., percutaneous nephrostomy tube, indwelling balloon catheter, bile duct drainage tube, or pleural / peritoneal / pericardial catheter) is allowed. Dedicated central infusion catheters, such as Port-a-Cath or Hickman catheters; 9) History or presence of detectable cerebrospinal fluid (CSF) malignant cells or brain metastases, unless otherwise specified in the substudy eligibility criteria; 10) History or presence of central nervous system (CNS) disorders, such as cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease or any autoimmune disease with CNS involvement, reversible posterior encephalopathy syndrome, or cerebral edema with structural defects confirmed by appropriate imaging. History of stroke or transient ischemic attack within 12 months prior to enrollment.Subjects with epileptic disorders requiring active anticonvulsant medication; 11) Presence of atrial or ventricular lymphoma involvement; 12) History of myocardial infarction, angioplasty or stenting, unstable angina, or other clinically significant cardiac disease within 12 months prior to enrollment; 13) Need for urgent treatment due to tumor mass effect (e.g., vascular compression, intestinal obstruction, or transmural gastric involvement); 14) Presence of primary immunodeficiency; 15) History of autoimmune disease (e.g., Crohn's disease, rheumatoid arthritis, systemic lupus) leading to end-organ damage or requiring systemic immunosuppression / systemic disease modulators within the past 2 years; 16) History of deep vein thrombosis or pulmonary embolism requiring therapeutic anticoagulation within 6 months prior to enrollment; 17) Any medical condition that may interfere with the safety or efficacy assessment of the study treatment; 18) History of severe immediate allergic reaction to any agent used in this study; 19) ≤ 5 years before the planned start of the lymphodepleting chemotherapy regimen 6 weeks of live vaccine and is expected to need this vaccine during the first 12 months after the infusion of Brekky; 20) Women who are pregnant or breastfeeding (due to the potential hazardous effects of preparative chemotherapy on the fetus or infant). Women who have undergone surgical sterilization or have been postmenopausal for at least 2 years are not considered to be of childbearing potential; 21) Unwilling to practice birth control from the time of consent until 6 months after the infusion of Brekky Orensay; and 22) In the judgment of the investigator, the subject is unlikely to complete all study-specific visits or procedures (including follow-up visits) or comply with the participation requirements of the study.

[0352] For WM, substudy-specific inclusion criteria included: clinicopathological diagnosis of WM; ≥2 prior therapies for WM, including BTKi and chemotherapy with disease progression or nonresponse; need for treatment per guideline; and measurable disease (IgM level >2x upper limit of normal). For WM, substudy-specific exclusion criteria included: allogeneic SCT; autologous SCT was allowed if 6 months had passed; and past history of CNS involvement (Bing-Neel syndrome), unless brain MRI and CSF were free of pathology.

[0353] Regarding Richter's transformation, substudy-specific inclusion criteria included: diagnosis of CLL based on the 2018 IWCLL criteria, histologically confirmed Richter's transformation to DLBCL subtype; at least 1 measurable site of disease based on the 2014 Lugano criteria; and R / R RT defined as 1 primary refractory disease or relapse after ≥1 line of chemotherapy. Regarding Richter's transformation, substudy-specific exclusion criteria included: allogeneic or autologous SCT <3 months before screening and / or <4 months before planned infusion of bregalen; and the presence of active graft-versus-host disease after a previous stem cell transplant.

[0354] For Burkitt lymphoma / leukemia, substudy-specific inclusion criteria included: histologically confirmed mature B-cell NHL Burkitt lymphoma / leukemia; R / R BL defined as 1 primary refractory disease or relapse after ≥1 line of chemotherapy (including anthracyclines); and measurable disease by radiographic criteria or isolated bone marrow involvement. For Burkitt lymphoma / leukemia, substudy-specific exclusion criteria included: allogeneic SCT <3 months prior to screening, and patients with active graft-versus-host disease prior to allogeneic stem cell transplant.

[0355] For hairy cell leukemia, substudy specific inclusion criteria included: histologically confirmed hairy cell leukemia; need for therapy based on: - neutrophils <1.0 × 10 9 / L, platelets <100×10 9 / L, hemoglobin <11 g / dL, and symptomatic splenomegaly or lymphadenopathy; and at least 2 prior systemic therapies, including at least one PNA and moxetumomab pasudotox (if eligible and available).

[0356] In certain aspects, bridging therapy can be administered after leukapheresis and before lymphodepleting conditioning chemotherapy. In certain other aspects, bridging therapy must be completed ≥ 7 days or 5 half-lives before lymphodepleting conditioning chemotherapy.

[0357] Subjects with Richter's 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 inhibitor (BTKi) (BTKi) ± VTX-2337; dexamethasone; and radiation, at the discretion of the healthcare provider. Bridging therapy regimens for subjects with Richter's transformation include those summarized in Table 10. The doses listed are embodiments only and may be adjusted based on age, comorbidities, or based on local or institutional guidelines.

[0358] Table 10. Bridging therapy regimens

[0359]

[0360] 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 with cyclophosphamide, doxorubicin, vincristine, and prednisolone; R-GEMOX, rituximab, gemcitabine, and oxaliplatin; R-ICE, rituximab, ifosfamide, carboplatin, and etoposide

[0361] Subjects with Burkitt's lymphoma may receive bridging therapy selected from the group consisting of: rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine, and prednisone (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 radiation, at the discretion of the healthcare provider.

[0362] Bridging therapy regimens for subjects with Burkitt's lymphoma include those outlined in Table 11. The doses listed are embodiments only and may be adjusted based on age, comorbidities, or according to local or institutional guidelines.

[0363] Table 11. Bridging therapy regimens

[0364]

[0365] Abbreviations: BID, twice daily; CLcr, 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 with gemcitabine and oxaliplatin; R-ICE, rituximab with ifosfamide, carboplatin, and etoposide.

[0366] Subjects with WM may receive bridging therapy with ibrutinib at the discretion of their healthcare provider.

[0367] Example 4

[0368] Brexu-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for relapsed / refractory mantle cell lymphoma (R / R MCL). After 3 years of follow-up in the pivotal Phase 2 ZUMA-2 study, 91% of MCL patients who progressed on BTK inhibitors responded to brexu-cel therapy, with a median duration of response (DOR) of 28.2 months. This example provides data evaluating patient, product, and PK characteristics in ZUMA-2 in progressive responders (patients who responded at the 24-month assessment), relapse responders (patients who initially responded but relapsed before the 24-month assessment), and non-responders.

[0369] At a median follow-up of 35.6 months, 68 patients received brexu-cel, with 91% of patients (n=62) achieving a response (CR or PR) and 9% of patients (n=6) not responding. Of the responders, 29 had an ongoing response (28 CRs and 1 PR) at their 24-month assessment (15 CRs and 14 PRs). The ongoing responders had a lower median baseline tumor burden (sum product diameter, 935 mm) than the relapsed responders. 2 Compare 4861mm 2 ) and a higher frequency of Eastern Cooperative Oncology Group (ECOG) performance status 0 (79% vs 59%). A smaller proportion of progressive responders received prior platinum therapy (10% vs 41%) or bridging therapy (21% vs 52%) compared with recurrent responders.

[0370] The median DOR was 47.1 months (95% CI, 36.5 months-not estimable) in progressive responders and 5.0 months (95% CI, 2.2-8.3) in recurrent responders. The median DOR was similar for patients with high baseline lactate dehydrogenase vs those with low baseline LDH, regardless of response status.

[0371] The median values ​​of peak and area under the curve for CAR T-cell expansion were approximately twice as high in progressive responders versus relapsed responders. Product characteristics were largely similar in progressive and relapsed responders, with a modest increase in the median total number of naive-like infused chemokine receptor 7 (CCR7)-positive T cells observed in progressive versus relapsed responders.

[0372] In summary, progressive responders had lower tumor burden, less use of prior platinum and bridging therapy, and higher CAR T cell expansion, suggesting that patients with lower overall disease burden or less prior chemotherapy may have a greater likelihood of durable response with brexu-cel. However, progressive responses were also observed in patients with high-risk disease features.

[0373] Introduction

[0374] Although Bruton's tyrosine kinase (BTK) inhibition has provided a paradigm shift in the treatment of mantle cell lymphoma (MCL), these agents have not yet proven to be effective. In patients with relapsed / refractory (R / R) MCL treated with BTK inhibitors, median progression-free survival (PFS) values ​​ranging from 13-33 months have been reported, and treatment interruptions due to progression or intolerance are common. The clinical benefit of BTK inhibitors is even more limited in patients with high-risk features, including first progression within 24 months of initial diagnosis (POD24), TP53 aberrations, elevated lactate dehydrogenase (LDH) at progression, and blastoid variants. In addition, outcomes after BTK inhibitor salvage therapy are poor, with reported median overall survival (OS) times as short as 2.5-8.4 months. In addition, the BTKi ibrutinib has recently been withdrawn from the MCL indication in the United States due to toxicity issues. Therefore, there remains an unmet need for better treatment options for patients with R / R MCL.

[0375] Chimeric antigen receptor (CAR) T-cell therapy represents another significant advance in the treatment of hematologic malignancies. Brexu-cel (formerly KTE-X19) is an autologous anti-CD19 CAR T-cell therapy approved in the U.S. for the treatment of adults with R / R MCL and in the EU for the treatment of adults with R / R MCL after ≥2 prior systemic therapies, including a BTK inhibitor. The accelerated approval was based on results from the pivotal, single-arm, multicenter Phase 2 ZUMA-2 (NCT02601313) study of brexu-cel therapy in patients with R / R MCL. All patients had progressed after BTK inhibitor therapy (62% refractory), and many had high-risk disease.

[0376] With a median follow-up of 35.6 months in ZUMA-2, 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) and was much longer in patients who achieved a CR (46.7 months) than in those with a partial response (PR; 2.2 months). The median PFS and OS were 25.8 months (95% CI, 9.6-47.6) and 46.6 months (95% CI, 24.9-not estimable [NE]), respectively. The most common grade ≥3 treatment-emergent adverse events (TEAEs) at a median follow-up of 12.3 months were cytopenias (94%) and infections (32%), with grade ≥3 cytokine release syndrome (CRS) and neurologic events occurring in 15% and 31% of patients, respectively. Similar real-world results were observed from the U.S. Lymphoma CAR T Consortium, which found that brexu-cel demonstrated an overall response rate of 90% and a complete response rate of 82% in 168 patients with R / R MCL who received brexu-cel in the standard-of-care setting. In addition, the study found that 8% and 32% of these patients experienced grade ≥3 CRS and neurologic toxicity, respectively, following brexu-cel infusion.

[0377] Understanding the association between patient, product, and pharmacokinetic characteristics and durable responses to brexu-cel in patients with R / R MCL may inform upfront patient selection to maximize benefit. This study examined the association of these factors with long-term responses to brexu-cel in ZUMA-2.

[0378] method

[0379] Study Design and Patients

[0380] Detailed methodology for the multicenter, single-group ZUMA-2 (NCT02601313) was described previously. Briefly, patients were ≥18 years of age and had histologically confirmed MCL that had relapsed after or was refractory to 1 to 5 prior MCL regimens, including anthracycline- or bendamustine-containing chemotherapy, anti-CD20 monoclonal antibodies, and BTK inhibitor therapy with ibrutinib or acalabrutinib. All patients underwent leukapheresis, after which patients with a high disease burden could receive bridging therapy with steroids or BTK inhibitors at the investigator’s discretion.

[0381] Conditioning chemotherapy consisted of intravenous (IV) fludarabine 30 mg / m2 once daily on days -5, -4, and -3. 2and cyclophosphamide 500 mg / m 2 On day 0, 2×10 6 A single IV infusion of brexu-cel was administered at a target dose of 10 CAR T cells / kg. All patients provided written informed consent, and the trial was conducted in accordance with the principles of the Declaration of Helsinki.

[0382] Endpoints and Assessments

[0383] This analysis examined baseline patient and disease characteristics, product characteristics, subsequent therapy, and pharmacological outcomes by response status at 24 months after brexu-cel infusion, as assessed by an independent radiology review committee using the Lugano classification. Progressive responders were defined as those with progressive CR or PR at their 24-month assessment. Relapsed responders were defined as those with a previous response who had relapsed, proceeded to subsequent anticancer therapy (including SCT), or died of any cause before their 24-month assessment. Nonresponders were patients who did not achieve a response. DOR was assessed in both responder groups. The levels of transduced anti-CD19 CAR T cells in the blood were measured by quantitative polymerase chain reaction. T cell phenotypes were assessed by multicolor flow cytometry using previously described protocols and antibodies.

[0384] Statistical analysis

[0385] All subgroup analyses were post hoc exploratory analyses, and descriptive statistics are provided. Time-to-event endpoints were analyzed using the Kaplan-Meier methodology.

[0386] result

[0387] patient

[0388] Of the 74 patients enrolled in ZUMA-2 who underwent leukapheresis, brexu-cel was successfully manufactured for 71 patients (95.9%), and 68 patients (91.9%) received brexu-cel. As of July 24, 2021, with a median follow-up of 35.6 months (range, 25.9-56.3), 62 patients (91.2%) achieved a best response of CR or PR, while 6 patients (8.8%) did not achieve a response. Of the 62 responders, 29 (47%; 28 CRs and 1 PR) were in an ongoing response at their 24-month assessment (ongoing responders), 29 (47%; 15 CRs and 14 PRs) relapsed before their 24-month assessment (relapsed responders), and 4 did not reach or missed their 24-month assessment and were excluded from this analysis.

[0389] Patient characteristics

[0390] Most baseline characteristics were similar between progressive responders, relapse responders, and nonresponders. However, progressive responders had approximately fourfold lower tumor burden at baseline compared with relapse responders (median SPD 935 mm 2 Compare 4861mm 2 ), were less likely to have received prior platinum therapy (10% vs 41%) or bridging therapy (21% vs 52%), were less likely to have POD24 (33% vs 66%), and were more likely to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 at baseline (79% vs 59%). Similar proportions of progressive and relapsed responders received prior bendamustine (45% vs 52%), prior anthracycline (76% vs 72%), and prior proteasome inhibitor (41% vs 34%), respectively. These trends generally held true when only progressive CR responders were compared with relapsed CR responders.

[0391] Across all subgroups, the median number of prior therapies was 3. Ibrutinib was the most common prior BTKi received across all subgroups and was the most common last prior therapy received across all subgroups; although it was more common in progressive responders (93% overall; 66% as last prior therapy) than in relapsed responders (79% overall; 41% as last prior therapy) or nonresponders (67% overall; 33% as last prior therapy). 28% of progressive responders, 21% of relapsed responders, and 33% of nonresponders received prior acalabrutinib; and it was the last prior therapy for 14% of progressive responders, 14% of relapsed responders, and 17% of nonresponders). The median time from the last prior therapy to brexu-cel infusion was 63 days (range, 26-748) for progressive responders, 63 days (range, 22-443) for relapsed responders, and 136 days (range, 29-642) for nonresponders.

[0392] effect

[0393] Among patients who achieved a CR, the median DOR was not achieved in progressive responders (n=28) (95% CI, 46.7-NE) and was 8.3 months (95% CI, 5.0-13.6) in relapsed responders (n=15). At the time of data collection, 22 of 28 progressive CR responders (79%) were in progressive response without subsequent therapy, 1 (4%) progressed to new anticancer therapy, 2 (7%) had disease progression, and 3 (11%) died. At the time of data collection, no responder of the 15 relapsed CR responders was in progressive response without subsequent therapy, 2 (13%) progressed to subsequent SCT, 1 (7%) progressed to new anticancer therapy, 12 (80%) had disease progression, and no responder died.

[0394] Among patients who achieved any response (CR or PR), the median DOR was 47.1 months (95% CI, 36.5-NE) in progressive 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 progressive 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 progressive 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 was associated with longer median DOR, PFS, and OS.

[0395] Considering that elevated LDH is a known adverse prognostic indicator for patients with R / R MCL, DOR was evaluated in patients with high and low baseline LDH. The median DOR in progressive responders 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 relapse 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.

[0396] One of 29 (3%), 20 of 29 (69%), and 3 of 6 (50%) patients received subsequent anticancer agents in the progressive responder, relapsed responder, and nonresponder subgroups, respectively. The most common of these therapies were radiation therapy, rituximab, dexamethasone, lenalidomide, and venetoclax.

[0397] Product and pharmacokinetic characteristics

[0398] Product characteristics were generally similar between progressive responders and relapsers. Numerical differences were observed between 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) for progressive responders, relapsers, and nonresponders, respectively. Although the total number of infused CAR T cells was similar between subgroups, the total number of infused CCR7+ T cells was modestly increased in progressive responders relative to the other subgroups, with a median level (x10 6 ) were 119.8 (range, 37.0-249.9), 89.4 (range, 6.1-353.4), and 88.2 (range, 39.9-150.3), respectively.

[0399] 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 progressive responders and relapsed responders, respectively. Similarly, the median CAR T-cell area under the curve (from day 0 to day 28; AUC 0-28 ) values ​​in these subgroups were 1487.0 cells / µl × day (range, 3.8-16700) and 775.8 cells / µl × day (range, 19.0-27200), respectively. Nonresponders had the lowest median CAR T cell peak (5.9 cells / µl; range, 0.2-95.9) and AUC of any subgroup. 0-28 value (24.7 cells / µl × number of days; range, 1.8-1089.1).

[0400] Differences in peripheral blood T cell phenotypes were observed between subgroups at day 7, including significantly higher median proportions of differentiated CD8+CD27-CD28+ cells and CD8+CCR7-CD45RA+CD27-CD28+ terminally differentiated effector memory cells in combined relapsers and nonresponders compared with progressive responders (P=0.0023 and P=0.0052, respectively; Table 12). Progressive responders had significantly more peripheral CD4+ T cells that maintained juvenile CD27+ expression than combined relapsers and nonresponders (P=0.03) and showed a trend toward higher levels of activated CD8 effector memory T cells (P=0.057; Table 12).

[0401] Table 12

[0402]

[0403]

[0404] discuss

[0405] This analysis identified associations between patient, disease, product, and / or pharmacokinetic characteristics and durable responses to brexu-cel in patients with R / R MCL treated in ZUMA-2. The analysis identified a 47% rate of progressive response at 24 months after infusion. Given the poor prognosis and limited survival associated with the failure of BTK inhibitors in this setting, these results continue to support brexu-cel as a favorable treatment option in this disease setting. Interestingly, the median DOR for relapsed responders was only 5 months, while for progressive responders it was 47.1 months, suggesting that patients who were still in progressive response after 24 months had favorable long-term outcomes. Notably, the significant differences in the Kaplan-Meier DOR curves for progressive responders versus relapsed responders between 6 and 12 months suggest that these earlier time points can predict long-term responses.

[0406] Because CAR T-cell therapy is a relatively recent approach to treating patients with hematological malignancies, factors associated with response are under investigation. These factors may be specific to a given CAR T-cell product and / or malignancy, and conclusive data are not yet available. Patient characteristics (such as baseline tumor burden and LDH levels) as well as T-cell phenotypes (such as the proportion of memory T cells and the CD4+ / CD8+ T-cell ratio) have been associated with response or durability of response to CAR T-cell therapy targeting CD-19. Recently, tumor immune organization has been implicated as a determinant of CAR T-cell efficacy and may also play a role in durability of response in this analysis.

[0407] In ZUMA-2, tumor burden measured by SPD at baseline was significantly lower in progressive responders than in relapsed responders (935 mm 2 Compare 4861mm 2). A similar correlation between SPD and outcome has been reported in B-cell malignancies in another study of CAR T-cell therapy. Prior platinum therapy and bridging therapy were less frequent in progressive responders than in relapsed responders, and progressive 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 is associated with a higher risk of relapse at 24 months. In contrast, the known risk factor of high baseline LDH levels (≥upper limit of normal) was not associated with worse response durability, as patients with low and high baseline LDH (progressive responders or relapsed responders) had similar median DoRs within each subgroup. Therefore, brexu-cel was associated with durable responses regardless of LDH status at baseline.

[0408] Ibrutinib was received as prior therapy and as the last prior therapy more frequently in progressive responders than in relapsed responders or nonresponders. Interestingly, the same phenomenon was not observed with prior acalabrutinib therapy, as similar proportions of progressive responders, relapsed responders, and nonresponders received acalabrutinib as prior therapy or as the last prior therapy. In preclinical studies, ibrutinib has been shown to improve CAR T-cell persistence and efficacy, possibly through its off-target inhibition of inducible T-cell kinases to improve T-cell function and expansion. Furthermore, in the TARMAC trial, the time-limited combination of ibrutinib and tesalencel showed encouraging efficacy (90% ORR) in a small cohort of patients (N=20) with R / R MCL. Additional clinical studies are needed to further understand the impact that prior ibrutinib may have on the long-term efficacy of brexu-cel therapy.

[0409] In a previous analysis of ZUMA-2, prior bendamustine use within 6 months of apheresis was associated with a worse pharmacokinetic profile and reduced product doubling time of infused CAR T cells, suggesting that the timing of bendamustine use could attenuate T-cell health; although the small sample size limits the interpretation of this finding. In the current analysis, the rates of prior bendamustine use were similar in ongoing responders and relapsing responders; however, due to the small sample size, it was not possible to assess whether the timing of prior bendamustine affected the durability of response.

[0410] It is reasonable to expect that CAR T products derived from patients with different T cell subsets will vary in their effects. In ZUMA-2, T cell phenotypic data were available for most patients, allowing comparison of T cell subsets in progressive responders versus combined relapsed responders / non-responders. The increase in the median number of CCR7+ cells and CD27+ peripheral T cells in progressive responders suggests that continuous memory T cell differentiation may play a role in achieving a durable response.

[0411] In a previous study of brexu-cel (ZUMA-1), peak CAR T cell levels and CAR T cell AUC were found to be associated with durable (24 months) responses. In patients with R / R MCL treated with brexu-cel in ZUMA-2, the present analysis identified similar trends. Both median peak and AUC levels of CAR T cells were approximately twofold higher in progressive responders, suggesting that robust CAR T cell expansion may contribute to achieving durable responses. These findings are consistent with the results of ZUMA-7, even though this effect was not observed in other CAR T studies using different CAR T products (e.g., JULIET). Factors specific to the disease context, tumor characteristics (including the microenvironment), and patient baseline characteristics as well as product characteristics preclude extrapolation or generalization.

[0412] These results demonstrate durable responses in patients with R / R MCL treated with brexu-cel, including those with high-risk disease features but otherwise a poor prognosis. Several factors that appeared to be associated with a 2-year progressive response (ie, ECOG performance status, SPD, prior platinum, use of bridging therapy) were related to the advanced or aggressive nature of the disease, suggesting that the optimal use of brexu-cel may be earlier in the disease. Consistent with some but not all previous CAR T studies, the extent of CAR T-cell expansion correlated with durability of response. These findings, along with those from further studies, will help identify patients who may derive the greatest benefit from brexu-cel in this difficult-to-treat malignancy.

[0413] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0414] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure.

Claims

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

2. The method of claim 1, wherein the subject has received 1 to 5 prior therapies, wherein at least one of the prior therapies is selected from autologous SCT, anti-CD20 antibodies, and / or chemotherapy containing anthracyclines or bendamustine.

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

4. The method of any one of claims 1 to 3, wherein R / RB cell ALL is defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≤12 months after first remission, relapsed or is refractory after ≥2 prior lines of systemic therapy, or relapsed after allogeneic SCT, wherein the subject is required to have ≥5% bone marrow blasts, an Eastern Cooperative Oncology Group performance status of 0 or 1, and / or adequate renal, hepatic, and cardiac function.

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

6. The method according to any one of claims 1 to 5, wherein the subject receives bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

7. The method according to any one of claims 1 to 6, wherein the MCL subject receives 500 mg / m 2 intravenously administered on each of the fifth, fourth, and third days prior to T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously 2 Fludarabine and both lymphodepleting chemotherapy regimens.

8. The method of any one of claims 1 to 7, wherein the B-cell ALL subject receives 25 mg / m per day of 1% paraformaldehyde administered intravenously (IV) on each of the fourth, third, and second days prior to T cell infusion. 2 Fludarabine plus 900 mg / m2 / day given IV on the day before infusion 2 Lymphodepletion regimen with cyclophosphamide.

9. The method of any one of claims 6 or 8, wherein the MCL bridging therapy is selected from dexamethasone (e.g., 20 mg to 40 mg or equivalent given PO or IV daily for 1 to 4 days); methylprednisolone, ibrutinib (e.g., 560 mg given PO daily) and / or acalabrutinib (e.g., 100 mg given PO twice daily); an immunomodulator; R-CHOP, bendamustine; an alkylating agent; and / or a platinum-based agent, wherein the bridging therapy is administered after leukapheresis and is completed 5 days or less prior to, e.g., conditioning chemotherapy.

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

11. The method of 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 subsequent partial or complete depletion of circulating cancer cells.

12. The method of claim 11, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective 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-ζ domains.

13. The method of claim 11 or 12, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

14. The method of any one of claims 11 to 13, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

15. The method of claim 14, wherein the superior product attribute is selected from increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production and / or decreased percentage of CD3- cells.

16. The method according to any one of claims 1 to 15, wherein 1.8×10 6 pcs, 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients 100 kg and above), and 0.5×10 6 pcs, 1×10 6 or 2×10 6 CAR-positive live T cells / kg body weight, with the maximum value of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

17. The method of any one of claims 1 to 15, wherein if the subject has achieved a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells if progression occurs after remission for >3 months, provided CD19 expression has been preserved and neutralizing antibodies against the CAR are not suspected, wherein the response is assessed using the Lugano classification.

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

19. The method of claim 18, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

20. The method of claim 18 or 19, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and / or hypotension, and signs or symptoms associated with neurological events include encephalopathy, seizures, changes in level of consciousness, speech disorders, tremors and / or confusion.

21. The method of any one of claims 18 to 20, wherein cytokine release syndrome in a subject with MCL is managed according to the following regimen: 。 22. The method of any one of claims 18 to 21, wherein neurotoxicity in a subject with MCL is managed according to the following regimen: 。 23. The method according to any one of claims 1 to 22, wherein the MCL subject is a high-risk patient determined by the presence of a Ki-67 tumor proliferation index ≥ 50% and / or a TP53 mutation.

24. The method of any one of claims 18 to 20, wherein CRS in a subject with B-cell ALL is managed according to the following regimen: 。 25. The method of any one of claims 18 to 20 and 24, wherein neurotoxicity in a subject with B-cell ALL is managed according to the following regimen: 。 26. The method according to any one of claims 1 to 25, wherein the B-cell ALL subject may receive any one or more of the following bridging chemotherapy regimens: 。 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. Use of autologous T cells expressing anti-CD19 CAR in the manufacture of a medicament 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 in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), wherein the MCL is relapsed or refractory MCL and the last prior therapy was less than 60 months prior to administration of the T cell product.

30. The method of claim 29, wherein the MCL is refractory to, or has relapsed following, 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.

31. The method of claim 29 or 30, wherein the subject has received 1 to 3 prior therapies, wherein at least one of the prior therapies is selected from autologous SCT, anti-CD20 antibodies, anthracycline- or bendamustine-containing chemotherapy, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

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

33. The method of claim 32, wherein ibrutinib is the last treatment prior to administration of the T cell product.

34. The method of any one of claims 29 to 33, wherein the subject has not received bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

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

36. The method of any one of claims 29 to 35, wherein the subject receives 500 mg / m 2 administered intravenously on each of the fifth, fourth, and third days prior to T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously 2 Fludarabine and both lymphodepleting chemotherapy regimens.

37. The method of 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 subsequent partial or complete depletion of circulating cancer cells.

38. The method of claim 37, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective 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-ζ domains.

39. The method of claim 37 or 38, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

40. The method of any one of claims 37 to 39, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

41. The method of claim 40, wherein the superior product attribute is selected from the group consisting of increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production, decreased percentage of CD3- cells.

42. The method of any one of claims 29 to 41, wherein 1.8 x 10 6 , 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

43. The method of any one of claims 29 to 42, wherein if the subject has achieved a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells if progression occurs after remission for >3 months, provided CD19 expression has been preserved and neutralizing antibodies against the CAR are not suspected, wherein the response is assessed using the Lugano classification.

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

45. The method of claim 44, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

46. ​​The method of any one of claims 44 and 45, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and / or hypotension, and signs or symptoms associated with neurotoxicity include encephalopathy, seizures, changes in level of consciousness, speech disorders, tremors and / or confusion.

47. The method of any one of claims 44 to 46, wherein cytokine release syndrome in a subject with MCL is managed according to the following regimen: 。 48. The method of any one of claims 44 to 47, wherein neurotoxicity in a subject with MCL is managed according to the following regimen: 。 49. The method according to any one of claims 29 to 48, wherein the subject is a high-risk patient determined by the presence of a Ki-67 tumor proliferation index ≥ 50% and / or 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 medicament 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 Waldenstrom's macroglobulinemia, Reye's transformation, Burkitt's lymphoma, and hairy cell leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), wherein the subject receives bridging therapy after leukapheresis and before conditioning chemotherapy / lymphodepleting chemotherapy.

53. The method of claim 52, wherein the cancer is refractory to, or has relapsed following, one or more of chemotherapy, radiation therapy, immunotherapy, autologous stem cell transplantation, or any combination thereof.

54. The method of claim 52 or 53, wherein the bridging therapy is completed for ≥7 days or ≥5 half-lives prior to the conditioning chemotherapy.

55. The method of any one of claims 52 to 54, wherein the subject receives 500 mg / m 2 administered intravenously on each of the fifth, fourth, and third days prior to T cell infusion. 2 Cyclophosphamide and 30 mg / m given intravenously 2 Fludarabine and both lymphodepleting chemotherapy regimens.

56. The method of any one of claims 52 to 55, wherein the cancer is Reye's 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 radiation.

57. The method of any one of claims 52 to 55, wherein the cancer is Burkitt's 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 radiation.

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

59. The method of 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 subsequent partial or complete depletion of circulating cancer cells.

60. The method of claim 59, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing the FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3-ζ domains.

61. The method of claim 59 or 60, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected for CD4+ and CD8+ T cells.

62. The method of any one of claims 59 to 61, wherein the T cell product has other superior product attributes relative to a T cell product comprising T cells from a leukapheresis-derived product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

63. The method of claim 62, wherein the superior product attribute is selected from the group consisting of increased percentage of CDRA45+CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN-γ production, decreased percentage of CD3- cells.

64. The method of any one of claims 52 to 63, wherein 1.8 x 10 6 , 1.9×10 6 or 2×10 6 One or more doses of CAR-positive live T cells / kg body weight, with a maximum of 2×10 8 CAR-positive live T cells (for patients weighing 100 kg and above).

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

66. The method of claim 65, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably for four weeks following infusion.

67. The method of claim 65 or 66, wherein signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and signs or symptoms associated with neurological events include encephalopathy, seizures, changes in 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 medicament for treating cancer according to any one of claims 52 to 67.

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

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

72. The method of claim 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 subsequent partial or complete depletion of circulating cancer cells.

73. The method of claim 72, wherein the CD4+ and CD8+ T cells have been activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and then transduced with a replication-defective viral vector encoding a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3-ζ domains.

74. The method of any one of claims 70 to 73, wherein the cancer is selected from the group consisting of mantle cell lymphoma (MCL), B-cell ALL, Waldenstrom's macroglobulinemia, Reye's transformation, Burkitt's lymphoma, and hairy cell leukemia.

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

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

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

78. The method of claim 77, wherein the blood sample is collected from the subject on day 7 after administration of the first T cell product.

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

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

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

82. The method of claim 70, wherein the subject's elevated CD8+CD27-CD28+ T cell levels are determined by comparison with other subjects who have received a comparable T cell product and whose peripheral blood samples were collected on the same day after administration of the T cell product.

83. The method of claim 71, wherein the subject's elevated CD8+CCR7-CD45RA+CD27-CD28+ T cell levels are determined by comparison with other subjects who have received a comparable T cell product and whose peripheral blood samples were collected on the same day after administration of the T cell product.

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

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 medicament for treating cancer according to any one of claims 70 to 84.

87. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising (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, and (c) prescribing a course of treatment 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, administering a second T cell product.

88. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising (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, and (c) prescribing a course of treatment 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, administering a second T cell product.

89. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising (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 said blood sample, and (c) prescribing a course of treatment based on the level of CD27+CD28- CD4+ CD3+ T cells in said blood sample, wherein if the level of CD27+CD28- CD4+ CD3+ T cells is elevated, then not administering a second T cell product.

90. A method for monitoring a subject who has previously been administered a first T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the method comprising (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, and (c) prescribing a course of treatment 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, then not administering a second T cell product.

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