Chimeric antigen receptor T cell therapy

Abstract

Provided herein are methods for preparing, producing, treating, culturing, isolating, or generating cells suitable for immunotherapy or cell therapy, and their use in cell therapy.

Description

[Technical Field]

[0001] This application relates to CAR-T cells, methods for their production, and methods for their use to treat cancer. [Background technology]

[0002] Human cancers, by their very nature, consist of normal cells that have undergone genetic or epigenetic alterations to become abnormal cancer cells. Cancer cells express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ 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 generate populations of chimeric antigen receptor (CAR) T cells for cancer therapy by preparing T cell populations enriched in naturally occurring T cells capable of targeting tumor antigens, depleting circulating tumor cells, and / or genetically modifying T cells to specifically target known cancer antigens. Some of these therapies have shown promising effects on tumor size and patient survival. Summary of the Invention

[0003] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. While the present invention has been described in conjunction with its detailed description, this description is intended to be illustrative and not limiting of the scope of the invention, which is defined in part by the appended claims. Other aspects, advantages, and modifications are within the scope of the following embodiments / claims.

[0004] Embodiment 1. A method in a subject in need of treatment for mantle cell lymphoma (MCL) or B-cell ALL, 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).

[0005] Embodiment 2. The method of embodiment 1, wherein the MCL and B-cell ALL is relapsed or refractory MCL and B-cell ALL, and optionally, the MCL is classical, blastoid, or pleomorphic MCL.

[0006] Embodiment 3. The method of any one of embodiments 1 and 2, wherein the MCL and B-cell ALL is refractory to or has relapsed after 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 transplant, or any combination thereof.

[0007] Embodiment 4. The method of any one of embodiments 1-3, wherein the subject has received 1 to 5 prior therapies, optionally at least one of the prior therapies is selected from autologous SCT, anti-CD20 antibodies, chemotherapy including anthracyclines or bendamustine, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

[0008] Embodiment 5. The method of embodiment 4, wherein the BTKi is ibrutinib or acalabrutinib.

[0009] Embodiment 6. The method of any one of embodiments 1-5, wherein R / RB cell ALL is defined as resistance to first-line therapy (i.e., primary therapy resistance), relapse within 12 months of first remission, relapse or resistance after two or more prior systemic therapies, or relapse after allogeneic stem cell transplant (SCT), and optionally, subjects are required to have bone marrow blasts ≥ 5%, Eastern Cooperative Oncology Group performance status of 0 or 1, and / or adequate renal, hepatic, and cardiac function.

[0010] Embodiment 7. The method of any one of embodiments 1-6, wherein if the subject with B-cell ALL has received prior blinatumomab, the subject is required to have leukemic blasts with CD19 expression > 90%.

[0011] Embodiment 8. The method of any one of embodiments 1-7, wherein the subject receives bridging therapy after leukapheresis and before conditioning / lymphocyte-depleting chemotherapy.

[0012] Embodiment 9. The method of any one of embodiments 1-8, wherein the subject with MCL receives a lymphodepleting chemotherapy regimen of intravenous cyclophosphamide 500 mg / m2 and intravenous fludarabine 30 mg / m2, both of which are administered 5 days, 4 days, and 3 days, respectively, prior to the T cell infusion.

[0013] Embodiment 10. The method of any one of embodiments 1-9, wherein the subject with B-cell ALL receives a lymphocyte-depleting regimen of intravenous (IV) fludarabine 25 mg / m2 / day 4, 3, and 2 days prior to each of the T-cell infusions, and IV cyclophosphamide 900 mg / m2 / day 2 days prior to the infusion.

[0014] Embodiment 11. The method of any one of embodiments 8-10, wherein the MCL bridging therapy is selected from dexamethasone (e.g., PO or IV 20-40 mg equivalent, once daily for 1-4 days); methylprednisolone, ibrutinib (e.g., PO 560 mg once daily), and / or acalabrutinib (e.g., PO 100 mg twice daily); immunomodulatory agents; R-CHOP, bendamustine; alkylating agents; and / or platinum-based agents, and wherein the bridging therapy is administered after leukapheresis, e.g., completed within 5 days prior to conditioning chemotherapy.

[0015] Embodiment 12. The method of any one of embodiments 8-10, wherein the subject with B-cell ALL may receive any one or more of the following bridging chemotherapy regimens: TIFF0007813701000001.tif83162

[0016] Embodiment 13. The method of any one of embodiments 1-12, 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.

[0017] Embodiment 14. The method of embodiment 13, wherein PBMCs are enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-deficient viral vector containing FMC63-28Z CAR, a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28, and a CD3 zeta domain.

[0018] Embodiment 15. The method of any one of embodiments 13 and 14, wherein the T cell product contains fewer cancer cells than a T cell product comprising T cells from a leukapheresis product that has not been positively selected for CD4+ and CD8+ T cells.

[0019] Embodiment 16. The method of any one of embodiments 13-15, wherein the T cell product has other superior product properties relative to T cell products comprising T cells from a leukapheresis product that have not been positively selected / enriched for CD4+ and CD8+ T cells.

[0020] Embodiment 17. The method of embodiment 16, wherein the superior product characteristics are selected from an increased percentage of CDRA45+CCR7+ (naive-like) T cells, a decreased percentage of differentiated T cells, an increased percentage of CD3+ cells, decreased IFN-γ production, and a decreased percentage of CD3- cells.

[0021] Embodiment 18. The method of any one of embodiments 1-17, wherein a subject with MCL is administered one or more doses of 1.8x106, 1.9x106, or 2x106 viable CAR-positive T cells per kg of body weight, up to a maximum of 2x108 viable CAR-positive T cells (for patients weighing 100 kg or more), and a subject with B-cell ALL is administered 0.5x106, 1x106, or 2x106 viable CAR-positive T cells per kg of body weight, up to a maximum of 2x108 viable CAR-positive T cells (for patients weighing 100 kg or more).

[0022] Embodiment 19. The method of any one of embodiments 1-17, wherein if the subject achieves a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells, and if the subject progresses after more than 3 months of remission, provided that CD19 expression is retained and neutralizing antibodies to the CAR are not suspected, and the response is assessed using the Lugano classification.

[0023] Embodiment 20. The method of any one of embodiments 1-19, wherein after administration of the T cells, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity.

[0024] Embodiment 21. The method of embodiment 20, wherein the subject is monitored for signs and symptoms of CRS and neurotoxicity for at least 7 days, preferably daily for 4 weeks, following the infusion.

[0025] Embodiment 22. The method of any one of embodiments 20 and 21, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and the signs or symptoms associated with a neurological event include encephalopathy, convulsions, altered level of consciousness, speech disturbance, tremor, and confusion.

[0026] Embodiment 23 The method of any one of embodiments 20-22, wherein the cytokine release syndrome in a subject with MCL is managed according to the following protocol: TIFF0007813701000002.tif140163

[0027] Embodiment 24. The method of any one of embodiments 20-23, wherein neurotoxicity in a subject with MCL is managed according to the following protocol: TIFF0007813701000003.tif123163

[0028] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the subject with MCL is a high-risk patient as determined by a Ki-67 tumor proliferation index of 50% or greater and / or the presence of a TP53 mutation.

[0029] Embodiment 26 The method of any one of embodiments 20-22, wherein CRS in a subject with B-cell ALL is managed according to the following protocol: TIFF0007813701000004.tif147160

[0030] Embodiment 27. The method of any one of embodiments 20-22 and 26, wherein neurotoxicity in a subject with B-cell ALL is managed according to one of the following two protocols: TIFF0007813701000005.tif234162

[0031] Embodiment 28. The method of any one of embodiments 1-27, wherein the subject with B-cell ALL may receive any one or more of the following bridging chemotherapy regimens: TIFF0007813701000006.tif84163

[0032] Embodiment 29. Autologous T cells expressing an anti-CD19 CAR for use in a method for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 28.

[0033] Embodiment 30. Use of autologous T cells expressing an anti-CD19 CAR in the manufacture of a medicament for treating mantle cell lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 28.

[0034] Embodiment 31. A prediction method comprising: (i) a method for predicting a subject's objective response rate to CAR T cell therapy (optionally according to the method of any one of embodiments 1 to 28), comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein the objective response rate is positively associated with the peak CAR T cell level, and the objective response rate includes both complete and partial responses, and all responses are assessed using the Lugano classification; (ii) a method for predicting minimal residual disease (e.g., at week 4) in response to CAR T cell therapy (optionally according to a method according to any one of embodiments 1 to 28), comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein negative minimal residual disease is associated with higher peak CAR T cell levels; (iii) a method for predicting grade ≧3 CRS and / or grade ≧3 neurological events (NE) in a subject who has received CAR T-cell therapy (optionally according to a method according to any one of embodiments 1 to 28), comprising measuring peak CAR T-cell proliferation after treatment and comparing the level with a reference value, wherein the higher the CAR T-cell proliferation, the higher the likelihood of grade ≧3 CRS and / or grade ≧3 NE events; (iv) a method for predicting grade ≧3 CRS and / or grade ≧3 NE, comprising measuring peak levels of GM-CSF and IL-6 after CAR T cell therapy (optionally according to a method described in any one of embodiments 1 to 28) and comparing them to reference levels, wherein the higher the peak levels of these cytokines, the higher the likelihood of grade ≧3 CRS and / or grade ≧3 NE; (v) a method for predicting Grade ≧3 CRS in a subject who has received CAR T-cell therapy (optionally according to a method described in any one of embodiments 1 to 28), comprising measuring the peak level of serum ferritin after CAR T-cell therapy and comparing it to a reference level, wherein the higher the peak level of ferritin, the higher the likelihood of Grade ≧3 CRS; (vi) A method for predicting grade ≧3 CRS, comprising measuring peak levels of serum IL-2 and IFN-γ after CAR T cell therapy (optionally as described in any one of embodiments 1 to 28) and comparing them to reference levels, wherein the higher the peak levels of IL-2 and IFN-γ, the higher the likelihood of grade ≧3 NE; (vii) A method for predicting Grade ≧3 CRS, comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecule (VCAM) after CAR T-cell therapy (optionally described in any one of embodiments 1 to 28) and comparing them to reference levels, wherein the higher the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecule (VCAM), the higher the likelihood of Grade ≧3 NE; (viii) A method of predicting Grade ≧3 CRS after CAR T cell therapy (optionally according to the method of any one of embodiments 1 to 28), comprising measuring peak serum levels of IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and / or perforin after anti-CD19 CAR T therapy and comparing the levels with reference levels, wherein peak serum levels of IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and / or perforin are positively associated with Grade ≧3 CRS; (ix) a method for predicting grade ≧3 CRS after CAR T-cell therapy of B-cell ALL (optionally according to the method of any one of embodiments 1 to 28), comprising measuring the peak serum level of IL-15 after anti-CD19 CAR T treatment and comparing the level to a reference level, wherein the peak serum level of IL-15 is negatively associated with grade ≧3 CRS; (x) a method for predicting grade ≧3 CRS and / or grade ≧3 NE after CAR T cell therapy (optionally according to the method of any one of embodiments 1 to 28), comprising measuring peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B after anti-CD19 CAR T therapy and comparing the levels with reference levels, wherein peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B are positively associated with grade ≧3 CRS and grade ≧3 NE; (xi) Patient achieves MRD (sensitivity 10-10) 4 weeks / 1 month after CAR T cell therapy (optionally described in any one of embodiments 1-28). -5 ) A method for predicting whether a patient will become MRD-negative at 1 month, comprising measuring peak serum levels of IFN-γ, IL-6, and / or IL-2 after treatment and comparing the levels to a reference standard, wherein peak serum levels of IFN-γ, IL-6, and / or IL-2 are positively associated with MRD negativity at 1 month.

[0035] Embodiment 32. The method of any one of embodiments 20-24, 26, 27, and 30-31, wherein CRS and NE are graded according to the method described in Lee et al., Blood 2014;124:188-195.

[0036] Embodiment 33. The method of embodiment 31, wherein the reference standard is established by any method commonly used in the biomarker field, such as quartile analysis of patient populations with known response, toxicity grade, and MRD level.

[0037] Embodiment 34. The method of embodiment 31, wherein the CAR T cell level is measured by the number of CAR gene copies per microgram of DNA in the blood.

[0038] Embodiment 35. The method of any one of embodiments 1-43, further comprising reducing the level / activity of cytokines positively associated with Grade ≥ 3 CRS and / or Grade ≥ 3 NE after CAR T cell infusion, thereby reducing Grade ≥ 3 CRS and / or Grade ≥ 3 NE.

[0039] Embodiment 36. A method of improving the efficacy of CAR T cell therapy (e.g., of classical, blastoid, and pleomorphic MCL, and B-cell ALL) in a subject in need thereof, comprising manipulating the T cell phenotype of a T cell product administered to the subject, optionally comprising increasing the number of CD3+ T cells, decreasing the number of CD3- cells, increasing the number / proportion of CDRA45+CCR7+ (naive-like) T cells and / or decreasing the number / proportion of differentiated cells in the T cell product during manufacture, decreasing the level of IFN-γ production by the T cells, wherein the improvement is observed relative to the efficacy of a T cell product prepared without any deliberate manipulation of the number / proportion of CDRA45+CCR7+ (naive-like) T cells and / or the number / proportion of differentiated cells in the T cell product. [Brief explanation of the drawings]

[0040] [Figure 1A] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53. [Figure 1B] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53. [Figure 1C] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53. [Figure 1D] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53. [Figure 1E] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53. [Figure 1F] Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and a trend toward increased cytokine levels in patients with mutant TP53.

[0041] [Figure 2A] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2B] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2C] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2D] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2E] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2F] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2G] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2H] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. [Figure 2I] Increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status.

[0042] [Figure 3] ZUMA-3 trial design. CAR, chimeric antigen receptor; DLT, dose-limiting toxicity.

[0043] [Figure 4] Diagram of ZUMA-3 CONSORT. *AEs were grade 3 pulmonary mass (n=1), grade 1 subdural hematoma (n=1), and grade 3 febrile neutropenia (n=1); †AEs were grade 4 sepsis (n=1) and grade 5 sepsis (n=1); ‡One patient received KTE-X19 off-label due to deep vein thrombosis, which was an exclusion criterion. AE, adverse event.

[0044] [Figure 5] Subgroup analysis of complete response rate. BM, bone marrow; ORR, overall remission rate; SCT, stem cell transplant.

[0045] [Figure 6] Duration of response, recurrence-free survival, and overall survival by dose.

[0046] [Figure 7] Association with peak CAR T cell proliferation and response, minimal residual disease, and toxicity.

[0047] [Figure 8] Association of CAR T-cell area under the curve with response, minimal residual disease, and toxicity. AE, adverse event; AUC, area under the curve; CAR, chimeric antigen receptor; CRS, cytokine release syndrome; MRD, minimal residual disease.

[0048] [Figure 9] Peak cytokine levels over time.

[0049] [Figure 10]Inflammatory markers in serum samples at baseline and peak post-infusion. *Values ​​represent the lower limit of quantification for the assay used. †Values ​​represent the upper limit of quantification for the assay used. AE, adverse event; CAR, chimeric antigen receptor; CCL, CC motif ligand; CRP, C-reactive protein; CXCL, CXC motif chemokine ligand; FGFBF, fibroblast growth factor basic form; FLT-1, fms-related receptor tyrosine kinase 1; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein-1; MDC, macrophage Phage-derived chemokine; MIP, macrophage inflammatory protein; PDL1, programmed death ligand 1; PLGF, placental growth factor; Rα, receptor α; RA, receptor antagonist; SAA, serum amyloid A; SFASL, soluble Fas ligand; TARC, thymus and activation-regulated cytokine; TNF, tumor necrosis factor; VCAM, vascular cell adhesion protein; VEGF, vascular endothelial growth factor; VEGFC, vascular endothelial growth factor C; VEGFD, vascular endothelial growth factor D.

[0050] [Figure 11] Association of serum biomarkers with cytokine release syndrome and neurological events. *Values ​​represent the lower limit of quantification for the assay used. †Values ​​represent the upper limit of quantification for the assay used. CRP, C-reactive protein; CXCL, CXC motif chemokine ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNγ, interferon gamma; IL, interleukin; IP, interferon gamma-inducible protein; MCP, monocyte-attractant protein; Rα, receptor alpha; RA, receptor antagonist; SAA, serum amyloid A.

[0051] [Figure 12]Pharmacodynamic profile of KTE-X19 across MCL morphological subgroups. AUC, area under the curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon-gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1β; PD-L1, programmed death ligand 1; PRF, perforin; Rα, receptor α; TNF-α, tumor necrosis factor α.

[0052] [Figure 13] Pharmacological profile of KTE-X19 across MCL morphological subgroups.

[0053] [Figure 14] Pharmacodynamic profile of KTE-X19 across BTKi-pretreated subgroups. AUC, area under the curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon-gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1β; PD-L1, programmed death ligand 1; PRF, perforin; Rα, receptor α; TNF-α, tumor necrosis factor α.

[0054] [Figure 15] Pharmacological profile of KTE-X19 across BTKi prior treatment subgroups.

[0055] [Figure 16] Ongoing response rates across subgroups. DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout the application. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. For example, "Concise Dictionary of Biomedicine and Molecular Biology" by Juo, Pei-Show, 2nd ed., 2002, CRC Press; "The Dictionary of Cell and Molecular Biology" 3rd ed., 1999, Academic Press; and "Oxford Dictionary of Biochemistry and Molecular Biology" Revised, 2000, Oxford University Press are provided to those skilled in the art as general dictionaries for many of the terms used in this application.

[0057] Units, prefixes, and symbols are presented in the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. The disclosure provided herein is not a limitation of various aspects of the present application, which may be incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 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, provide those skilled in the art with a comprehensive dictionary of many of the terms used in this disclosure.

[0058] The article "a" or "an" refers to "one or more" of any cited or listed components.

[0059] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend to some extent on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within a standard deviation of 1× or more, as practiced in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%). In the context of biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is presented in this application, unless otherwise specified, the meaning of "about" or "essentially comprising" includes an acceptable error range for that value or composition. Any concentration range, percentage range, ratio range, or integer range includes any integer value within the recited range, and fractions thereof, as appropriate (such as 1 / 10 and 1 / 100 of an integer), unless otherwise stated.

[0060] As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive and encompasses both "or" and "and." The term "and / or" refers to each of the two specified features or components, with or without the other. Thus, for example, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: 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).

[0061] The terms "for example" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to items explicitly listed herein.

[0062] Terms such as "more than," "at least," and "greater than," e.g., "at least one," are used to mean, 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, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, This includes 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or any value greater than the stated value. Any greater number or fraction in between is also included. The term "less than or equal to" includes each value less than the stated value.For example, "100 or fewer nucleotides" includes the following: 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 55 Included are 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0063] Terms such as "plurality," "at least two," "two or more," and "at least a second" are intended to mean, 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 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 , 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, including any larger number or fraction in between.

[0064] Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. Whenever an embodiment is described herein with the term "comprising," it will be understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also presented. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. In re Gray, 53 F.2d 520, 11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448,450 (Bd.App. 1948) ("consisting of" is defined as "closing a claim to include materials other than those recited, excluding impurities in natural association."). The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those "which do not materially affect the basic and novel characteristics" of the claimed patent.

[0065] As used herein, unless specifically stated otherwise or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one 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, "about" or "approximately" can mean within a standard deviation of 1× or more, as practiced in the art. "About" or "approximately" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean a difference of up to an order of magnitude or a difference of up to 5-fold. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition.

[0066] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, as appropriate (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0067] The terms "activation," "activated," and the like refer to a state of cells, including but not limited to immune cells (e.g., T cells), that have been stimulated sufficiently to induce detectable cell proliferation. Activation can be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell differentiation. 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 expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and International Publication No. WO 2012 / 079000, the entire contents of which are incorporated herein by reference. Generally, such methods involve contacting cells (such as T cells) with activators, stimulators, or costimulators (such as anti-CD3 and / or anti-CD28 antibodies), which may be attached, coated, or bound to beads or other surfaces, in a solution (such as feed medium, culture medium, and / or growth medium) containing specific cytokines (such as IL-2, IL-7, and / or IL-15). Activators (such as anti-CD3 and / or anti-CD28 antibodies) attached to the same beads function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In one embodiment, T cells are activated and stimulated to proliferate with specific antibodies and / or cytokines using methods described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and WO 2012 / 129514, the contents of which are incorporated herein by reference in their entireties.

[0068] The terms "administration," "administering," and the like refer to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of immune cells prepared by the methods disclosed herein include, for example, intravenous (iv or IV), intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. Parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In one embodiment, immune cells (e.g., T cells) prepared by the methods are administered via injection or infusion. Non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, twice, or multiple times over one or more periods of time. When more than one therapeutic agent (e.g., cells) are administered, administration can be simultaneous or sequential. Sequential administration involves administering one agent only after administration of one or more other agents is completed.

[0069] The term "antibody" (Ab) includes, but is not limited to, an immunoglobulin that specifically binds to an antigen. Generally, antibodies 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 can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass encoded by the heavy chain constant region gene (e.g., IgM or IgG1). The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human 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. Unless expressly stated, and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions, monovalent and divalent fragments or portions, of any of the foregoing immunoglobulins, and single-chain antibodies.

[0070] "Antigen-binding molecule," "antibody fragment," and the like refer to any portion of an antibody that is smaller than the whole. An antigen-binding molecule may include antigen-complementarity 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 from antigen-binding molecules. In one embodiment, a CD19 CAR construct comprises an anti-CD19 single-chain Fv. A "single-chain Fv" or "scFv" antibody-binding fragment comprises the variable heavy chain (V) of an antibody. H ) domain and variable light chain (V L ) domains, which are present in a single polypeptide chain. Generally, an Fv polypeptide comprises a V H Domains and V L The scFv further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. All antibody-related terms used herein have their conventional meaning in the art and are well understood by those of skill in the art.

[0071] "Antigen" refers to any molecule that can elicit an immune response or be bound by an antibody or antigen-binding molecule. The immune response can include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Antigens can be expressed endogenously, i.e., by genomic DNA, or recombinantly. Antigens can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Furthermore, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0072] The term "neutralizing" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or otherwise alters the ability of the ligand to bind through indirect means (such as a structural or energetic change in the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0073] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the methods of engineered autologous cell therapy described herein involve the collection of lymphocytes from an individual (such as a donor or patient), which are then engineered to express a CAR construct and then administered to the same individual.

[0074] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0075] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors that can invade neighboring tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors of various stages. In one embodiment, the cancer or tumor is at Stage 0, e.g., the cancer or tumor is in a very early stage of development and has not metastasized. In another embodiment, the cancer or tumor is at Stage I, e.g., the cancer or tumor is relatively small, has not spread to nearby tissues, and has not metastasized. In other embodiments, the cancer or tumor is at Stage II or Stage III, e.g., the cancer or tumor is larger than at Stage 0 or Stage I and has grown into neighboring tissues but has not metastasized, except possibly to the lymph nodes. In additional embodiments, the cancer or tumor is at Stage IV, e.g., the cancer or tumor has metastasized. Stage IV may also be referred to as advanced or metastatic cancer.

[0076] As used herein, "anti-tumor effect" refers to a biological effect that may be expressed, but is not limited to, as 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 survival or progression-free survival, an increase in life expectancy, and / or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect may also refer to the prevention of tumor development, e.g., vaccination.

[0077] The term "progression-free survival" (PFS) refers to the time from the date of treatment to the date of disease progression (according to common guidelines, such as the revised IWG response criteria for malignant lymphoma) or death from any cause. The term "disease progression" may be assessed by measuring 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 may be reported as the primary tumor type (e.g., DLBCL). The term "duration of response" (DOR) refers to the time from the subject's first objective response to the date of confirmed disease progression (according to common 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.

[0078] "Cytokine" refers to a non-antibody protein that can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. In one embodiment, one or more cytokines are released in response to a treatment. In other embodiments, the secretion of these cytokines in response to a treatment can indicate or suggest an effective treatment. In one embodiment, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell and mediates a response in the second cell. As used herein, "cytokine" refers to a protein released by one 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, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effector cytokines, and acute-phase proteins. For example, homeostatic cytokines such as interleukin (IL) 7 and IL-15 can promote immune cell survival and proliferation, while 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) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF).Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0079] A "chemokine" is a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0080] "Therapeutically effective amount," "therapeutically effective dose," and the like refer to the amount of cells (e.g., immune cells or engineered T cells) produced by the present method (resulting in a T cell product) that, when used alone or in combination with another therapeutic agent, protects or treats a subject against developing disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, and / or prevention of impairment or disability due to disease affliction. The ability to promote disease regression can be assessed using a variety of methods known to those of skill in the art, for example, by assaying the activity of an agent in a subject undergoing clinical trials, in an animal model system predictive of efficacy in humans, or in an in vitro assay. In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject who is not the patient. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells is at least about 10 4cells, 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 can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 In some embodiments, the therapeutically effective amount of viable CAR-positive T cells is about 1 x 10 cells / kg of body weight. 6 ~about 2×10 6 CAR-positive viable T cells, the maximum dose is approximately 1 x 10 8 In some embodiments, the therapeutically effective amount of CAR-positive viable T cells is about 0.4 x 10 8 ~about 2×10 8 In some embodiments, the therapeutically effective amount of CAR-positive viable T cells is about 0.4 x 10 8 , about 0.5×10 8 , about 0.6×108 , about 0.7×10 8 , about 0.8×10 8 , about 0.9×10 8 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , or approximately 2.0 × 10 8 These are CAR-positive viable T cells.

[0081] 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 (cytotoxic) lymphocyte that represents a major component of the genetic immune system. NK cells reject virus-infected tumors and cells through the process of apoptosis, or programmed cell death. They are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells.

[0082] 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 (TILs), myeloid-derived suppressor cells (MDSCs), and dendritic cells. The term also includes precursors of these immune cells. Hematopoietic stem 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 progenitor cells can differentiate into lymphoid, e.g., lymphoid, hematopoietic stem or progenitor cells. Additional examples of immune cells that can be used for immunotherapy are described in U.S. Patent Application Publication No. 2018 / 0273601, which is incorporated herein by reference in its entirety.

[0083] Several types of T cells exist, namely, helper T cells (e.g., CD4+ cells), effector T cells, EFF cell), cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell or killer T cell), memory T cell ((i) stem memory T cell) SCM The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, similar to naive cells, but also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells.), (ii) central memory T CM The cells express L-selectin and CCR7 + and CD45RO + and secrete IL-2 but not IFNγ or IL-4; and (iii) except that effector memory T EMThere are also T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells) that do not express L-selectin or CCR7 but do express CD45RO and produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. T cells found within tumors are called "tumor-infiltrating lymphocytes" (TILs). On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, from which they get their name.

[0084] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells become CD4 + or CD8 + In their naive state, T cells express L-selectin (CD62L + ), IL-7 receptor-α (IL-7R-α), and CD132, but do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" also refers to T cells that exhibit phenotypic characteristics of either naive or immature T cells, e.g., T SCM Cells or T CM For example, immature T cells express L-selectin (CD62L + ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive or immature T cells are T EM Cells and T EFF These may be contrasted with terminally differentiated effector T cells, such as T cells.

[0085] "T cell function," as referred to herein, refers to a normal characteristic of healthy T cells. T cell function can include T cell proliferation, T cell activity, and / or cytolytic activity. In one embodiment, the method of the present application of preparing T cells under specific oxygen and / or pressure conditions increases one or more T cell functions, thereby making the T cells more suitable and / or more potent for therapeutic purposes. In some embodiments, T cells prepared according to the method have increased T cell function compared to those under conditions lacking the specific oxygen and / or pressure. In other embodiments, T cells prepared according to the method may have increased T cell proliferation compared to T cells cultured under conditions lacking the specific oxygen and / or pressure. In additional embodiments, T cells prepared according to the method have increased T cell activity compared to T cells cultured under conditions lacking the specific oxygen and / or pressure. In further embodiments, T cells prepared according to the method have increased cytolytic activity compared to T cells cultured under conditions lacking the specific oxygen and / or pressure.

[0086] The terms "proliferation," "proliferating," and the like, of cells refer to the ability of cells to increase in number through cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation can occur in vitro, e.g., during T cell culture, or in vivo, e.g., after administration of 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 cell count (TVC). VCD or TVC can be a theoretical value (an aliquot or sample is removed from the culture at a specific time point, the cell number is determined, and then the cell number is multiplied by the culture volume at the start of the test) or an actual value (an aliquot or sample is removed from the culture at a specific time point, the cell number is determined, and then the cell number is multiplied by the actual culture volume at the specific time point). The term "T cell activity" refers to any activity common to healthy T cells. In one embodiment, T cell activity includes cytokine production (e.g., INFγ, IL-2, and / or TNFα). In another embodiment, T cell activity includes 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," and the like refer to the ability of a T cell to destroy a target cell. In one embodiment, the target cell is a cancer cell, e.g., a tumor cell. In another embodiment, the T cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cell expresses a target antigen.

[0087] The terms "genetically engineered," "gene editing," or "engineered" refer to methods of modifying a cell's genome, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In one embodiment, the modified cell is a lymphocyte, e.g., a T cell, and can be obtained from either a patient or a donor. The cell can be modified to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is integrated into the cell's genome.

[0088] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0089] The chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs) of the present application are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, such as T cells, according to techniques known in the art. In a CAR, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate the immune cell to attack and destroy cells bearing or expressing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. In one embodiment, the cells prepared according to the present application are cells bearing a chimeric antigen receptor (CAR) or a T cell receptor comprising an antigen-binding molecule, a costimulatory domain, and an activation domain. The costimulatory domain can comprise an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain comprises a hinge or truncated hinge domain.

[0090] "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 (including Abs, cytokines, and complement) produced either by these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0091] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or recurring with a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T cell and NK cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy, and allogeneic T cell transplantation. One skilled in the art will appreciate that the immune cell preparation methods disclosed herein enhance the efficacy of any cancer or transplant T cell therapy. Examples of T cell therapy are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent Nos. 7,741,465, 6,319,494, and 5,728,388, and WO 2008 / 081035, the contents of which are incorporated by reference herein in their entireties.

[0092] The term "engineered autologous cell therapy," also known as adoptive cell transfer and abbreviated as "eACT™," is the process of harvesting a patient's own T cells and subsequently genetically modifying them to recognize and target one or more antigens expressed on the 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 fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety containing a costimulatory domain and an activation domain.Examples of costimulatory domains include 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 costimulatory factor (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, and ICA. M-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp 46, CD19, CD4, CD8, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domain may be derived from 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 may be derived from CD3, such as CD3ζ, ε, δ, or γ.In one embodiment, the CAR is designed to have two, three, four, or more costimulatory domains. CAR scFvs can be designed to target, for example, CD19, a transmembrane protein expressed by cells of 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 Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties.

[0093] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or upregulation or downregulation of key molecules.

[0094] As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of a costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, and differentiation. Costimulatory ligands induce signals, for example, by binding of the T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule, in addition to the primary signal provided by the stimulatory molecule. Costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds 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. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory 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).

[0095] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecules" that are cognate binding partners on a T cell that specifically bind to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, and CD 45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19 a, 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), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, 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 (CD11a) / 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, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0096] In some embodiments, the cells of the present application can be obtained via T cells obtained from a subject. In one embodiment, T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with any solution (e.g., a solution with a neutral pH or PBS) or culture medium. As will be appreciated, the washing step can be performed, for example, using a semi-automated flow-through centrifuge, such as a Cobe™ 2991 cell processor, a Baxter CytoMate™, or the like. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions, with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0097] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation through a PERCOLL™ gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and / or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate a cell population of interest for use in the present disclosure.

[0098] In one embodiment, CD3+ T cells are isolated from PBMCs using Dynabeads coated with anti-CD3 antibodies, and CD8+ and CD4+ T cells are further isolated separately by positive selection using CD8 microbeads (e.g., Miltenyi Biotec) or CD4 microbeads (e.g., Miltenyi Biotec).

[0099] In some embodiments, PBMCs are used directly for genetic modification of immune cells (such as CARs) using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations before or after genetic modification and / or expansion.

[0100] The one or more immune cells described herein can be obtained from any source, including, for example, a human donor. The donor can be a subject in need of anti-cancer treatment, e.g., treatment with one of the immune cells generated by the methods described herein (i.e., an autologous donor), or can be an individual who provides a lymphocyte sample used to treat another individual or cancer patient upon generation of a cell population generated by the methods described herein (i.e., an allogeneic donor). The immune cells can be differentiated in vitro from a hematopoietic stem cell population, or the immune cells can be obtained from a donor. The population of immune cells can be obtained from a donor by any suitable method used in the art. For example, a population of lymphocytes can be obtained by any suitable extracorporeal method, venipuncture, or other blood collection method that obtains a blood sample with or without lymphocytes. The population of lymphocytes can be obtained by apheresis. The one or more immune cells can be harvested from any tissue containing one or more immune cells, including, but not limited to, a tumor. A tumor or a portion thereof is harvested from a subject, and one or more immune cells are isolated from the tumor tissue. Any T cells, including any immune cells suitable for T cell therapy, can be used in the methods disclosed herein. For example, one or more cells useful in the present application can be selected from the group consisting of tumor-infiltrating lymphocytes (TILs), cytotoxic T cells, CAR T cells, engineered TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a blood unit collected from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system, in which the human thymic environment is replicated to support efficient ex vivo differentiation of T cells derived from primary and reprogrammed pluripotent stem cells.Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication Nos. 2013 / 0287748, WO 2015 / 120096, and WO 2017 / 070395, all of which are incorporated by reference in their entireties for purposes of describing these methods. In one embodiment, the T cells are tumor-infiltrating leukocytes. In certain embodiments, one or more T cells express CD8, e.g., CD8. + In other embodiments, one or more T cells express CD4, e.g., CD4 + Further methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication Nos. 2013 / 0287748, WO 2015 / 120096, and WO 2017 / 070395, all of which are incorporated by reference in their entirety for purposes of describing these methods.

[0101] Immune cells and their progenitors can be isolated by available methods (see, e.g., Rowland-Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York (1999)). Sources of immune cells or their progenitors 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. Additionally, positive selection methods can isolate or enrich for the desired immune cells or their progenitors, or a combination of positive and negative selection methods may be used. Monoclonal antibodies (MAbs) are useful in both positive and negative selection to identify markers associated with specific cell lineages and / or stages of differentiation. When isolating a particular type of cell, e.g., a particular type of T cell, various cell surface markers or combinations of markers can be used to separate the cells, as is well known in the art, including, but not limited to, CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells) (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa NJ (2000); De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009)).

[0102] PBMCs can be used directly for genetic modification with immune cells (such as CARs). After isolating PBMCs, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into subpopulations of naive, memory, and effector T cells before or after genetic modification and / or expansion. In one embodiment, CD8+ cells can be further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In other embodiments, 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 certain embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In additional embodiments, CD4+ T cells can be further classified into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations bearing cell surface antigens.

[0103] The methods described herein further include enriching or preparing a population of immune cells obtained from the donor between collection from the donor and exposure to one or more cells obtained from the donor subject. Enrichment of a population of immune cells, e.g., one or more T cells, can be achieved by any suitable separation method, including, but not limited to, use of separation media (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical, Catalog No. 0850494X), cell size, shape, or density separation by filtration or elutriation, immunomagnetic separation (e.g., Magnetic Activated Cell Sorting System, MACS), fluorescent separation (e.g., Fluorescence Activated Cell Sorting System, FACS), or bead-based column separation.

[0104] In one embodiment, the T cells are obtained from a donor subject. In another embodiment, the donor subject is a human patient suffering from cancer or a tumor. In an additional embodiment, the donor subject is a human patient not suffering from cancer or a tumor. The present application also provides compositions or formulations comprising a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the composition or formulation comprises an excipient. The terms "crude" and "formulation" are used interchangeably herein. The terms composition, therapeutic composition, therapeutically effective composition, pharmaceutical composition, pharmaceutically effective composition, and pharmaceutically acceptable composition are used interchangeably herein. The composition may be selected for delivery via the digestive tract, such as parenteral delivery, inhalation, or oral administration. The composition may be prepared by methods known to those skilled in the art. A buffer is used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8. When parenteral administration is intended, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. For example, a parenteral injection vehicle is sterile distilled water in which the composition described herein, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. Preparations include formulating the desired agent with beads or liposomes, which are polymeric compounds (such as polylactic acid or polyglycolic acid) that allow for controlled or sustained release of the product, and then delivering them via depot injection. Additionally, implantable drug delivery devices can be used to introduce the desired therapeutic agent.

[0105] In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject that is not a patient. T cells 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 107 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 can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 In some embodiments, the therapeutically effective amount of viable CAR-positive T cells is about 1 x 10 cells / kg of body weight. 6 ~about 2×10 6 CAR-positive viable T cells, the maximum dose is approximately 1 x 10 8 These are CAR-positive viable T cells.

[0106] As used herein, a "patient" includes any human suffering from a disease or disorder, such as cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject," as used herein, refers to a subject from whom cells are being obtained for further in vitro manipulation. A donor subject may be a cancer patient (i.e., an autologous donor) who is treated with a population of cells generated by the methods described herein, or may be an individual who provides a lymphocyte sample used to treat another individual or cancer patient upon generation of a cell population generated by the methods described herein (i.e., an allogeneic donor). A subject who receives cells prepared by the methods may be referred to as a "recipient subject."

[0107] The terms "stimulation," "stimulating," and the like refer to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, which 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 present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an artificial antigen-presenting cell (aAPC), a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, such as, but not limited to, activation, initiation of an immune response, or proliferation. Stimulatory ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. As used herein, "activation" or "activity" refers to a T cell that has been stimulated. Activated T cells may be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

[0108] The term "exogenous activator" refers to any activator derived from an external source. For example, exogenous anti-CD3 antibody, anti-CD28 antibody, IL-2, exogenous IL-7, or exogenous IL-15 can be commercially obtained or recombinantly produced. When added to or contacted with one or more T cells, "exogenous IL-2," "exogenous IL-7," or "exogenous IL-15" indicates that such IL-2, IL-7, and / or IL-15 is not produced by the T cells. T cells prior to being mixed with "exogenous" IL-2, IL-7, or IL-15 may contain trace amounts produced by the T cells or isolated from a subject with the T cells (i.e., endogenous, "exogenous" IL-2, IL-7, or IL-15). One or more T cells described herein can be contacted with exogenous anti-CD3 antibody, anti-CD28 antibody, "exogenous" IL-2, IL-7, and / or IL-15 by any means known in the art, including adding isolated "exogenous" IL-2, IL-7, and / or IL-15 to the culture, adding anti-CD3 antibody, anti-CD28 antibody, "exogenous" IL-2, IL-7, and / or IL-15 to 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.

[0109] As used herein, the term "in vitro cells" refers to any cells cultured ex vivo. In one embodiment, in vitro cells include T cells.

[0110] The term "persistence" refers to the ability of, for example, one or more transplanted immune cells administered to a subject, or their progeny (e.g., differentiated or mature T cells), to remain detectably within the subject for a period of time. As used herein, increasing the persistence of one or more transplanted immune cells or their progeny (e.g., differentiated or mature T cells) refers to extending the period during which the transplanted immune cells are detectable in the subject following administration. For example, the in vivo persistence of one or more transplanted immune cells may be extended by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. Additionally, the persistence of the one or more transplanted immune cells in vivo may be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to one or more transplanted immune cells not prepared by the methods disclosed herein.

[0111] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change that is less than the original. "Reduce" and "reduce" are relative terms and require a comparison of measurements before and after. "Reduce" and "reduce" include complete depletion. The term "modulate" T cell maturation, as used herein, refers to the use of any of the interventions described herein to control the maturation and / or differentiation of one or more cells, such as T cells. For example, modulation refers to inactivating, delaying, or inhibiting T cell maturation. In another example, modulation 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" refers to maintaining T cells in a naive or T CM It can refer to maintaining the state, T EM or T EFF In addition, "delayed or inhibited T cell maturation" refers to the progression of immature or undifferentiated T cells (e.g., naive T cells and / or T CM The state of T cells (e.g., mature or immature) can be determined, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of T cells. For example, the presence of one or more markers selected from the group consisting of L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof, can indicate less mature, less differentiated T cells.

[0112] "Treatment" or "treating" 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 according to the present application to a subject / patient, with the intent of ameliorating, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, exacerbation, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In one aspect, "treatment" or "treating" includes partial remission. In another aspect, "treatment" or "treating" includes complete remission.

[0113] Various aspects of the application are described in further detail in the following subsections.

[0114] Patients with B-cell malignancies who have high levels of circulating CD19-expressing tumor cells represent a population with significant unmet need. For example, mantle cell lymphoma (MCL) is difficult to treat in the relapsed or refractory state and remains incurable. There is no standard of care for second-line and more advanced chemotherapy. Treatment options include cytotoxic chemotherapy, proteasome inhibitors, immunomodulatory agents, tyrosine kinase inhibitors, and stem cell transplantation (autologous [ASCT] and allogeneic stem cell transplantation [allo-SCT]). The choice of regimen is influenced by prior therapy, comorbidities, and tumor chemotherapeutic sensitivity. Despite high initial response rates observed with Bruton's tyrosine kinase inhibitors (BTK inhibitors), most patients ultimately develop progressive disease. Novel treatment strategies are needed to improve the poor prognosis of patients with relapsed / relapsed MCL that is not effectively controlled by chemoimmunotherapy, stem cell transplantation, and BTK inhibitors.

[0115] Products used in anti-CD19 CAR T-cell therapy, or CD19 CAR-T, can be manufactured from a patient's own T cells via leukapheresis, suitable for B-cell malignancies with circulating tumor cell burden, to minimize CD19-expressing tumor cells in the final product. T cells derived from leukocytes collected from the leukapheresis product can be enriched by selection of CD4+ / CD8+ T cells, activated with anti-CD3 and anti-CD28 antibodies, and / or transduced with a viral vector containing an anti-CD19 CAR gene. Further details of this method can be found in International Application No. US2015 / 014520, published as WO2015 / 120096, and International Application No. 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 expanded to generate sufficient numbers of cells to achieve a therapeutic effect. Such a process may eliminate malignant and normal B cells expressing CD19, reducing the activation, proliferation, and depletion of anti-CD19 CAR T cells.

[0116] The activation, transduction, and / or expansion of immune cells can be carried out for any suitable period of time that allows for the production of (i) a sufficient number of cells in a population of engineered immune cells for administration to a patient in at least one dose, (ii) a population of engineered immune cells with a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). The suitable period of time can depend on several parameters, including one or more populations of cells, cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and / or other variables. The activation period 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. The activation period according to the methods of the present application is shortened compared to expansion methods known in the art. For example, the period of activation may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. Furthermore, the period of proliferation may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more than 21 days. The period of proliferation according to the methods of the present application is reduced compared to proliferation methods known in the art. For example, the period of expansion may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. In one embodiment, the period of cell expansion is about 3 days and the period from enrichment of the cell population to generation of the engineered immune cells is about 6 days.

[0117] 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 can be 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 is determined by the expression of L-selectin (CD62L + ), IL-7Rα, and CD132. One skilled in the art will recognize that the present methods may increase the relative proportion of immature and undifferentiated T cells or DC cells in a population of collected cells, although some mature and differentiated cells may still be present. As a result, 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 a cell population before and after exposing one or more cells obtained from a subject to hypoxic culture conditions in the presence or absence of pressure above atmospheric pressure. The methods disclosed herein may increase the percentage of immature and undifferentiated T cells in a T cell population.

[0118] The methods described herein further include stimulating a population of cells, such as lymphocytes, with one or more T cell stimulatory agents to generate a population of activated T cells under appropriate conditions. The population of activated T cells can be generated using any combination of one or more suitable T cell stimulatory agents, including, but not limited to, antibodies or functional fragments thereof that target T cell stimulatory molecules or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies (e.g., OKT-3), anti-CD28 antibodies, or functional fragments thereof), or any other suitable mitogens (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands for T cell stimulatory molecules or costimulatory molecules.

[0119] Suitable conditions for stimulating or activating immune cell populations described herein further include temperature for a period of time and / or in the presence of a level of CO. The temperature for stimulation can be about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34-38°C, about 35-37°C, about 36-38°C, about 36-37°C, or about 37°C.

[0120] Other conditions for stimulating or activating immune cell populations described herein may further include a stimulation or activation time. The stimulation time is about 24 to 72 hours, about 24 to 36 hours, about 30 to 42 hours, about 36 to 48 hours, about 40 to 52 hours, about 42 to 54 hours, about 44 to 56 hours, about 46 to 58 hours, about 48 to 60 hours, about 54 to 66 hours, about 60 to 72 hours, about 44 to 52 hours, about 40 to 44 hours, about 40 to 48 hours, about 40 to 52 hours, or about 40 to 56 hours. In one embodiment, the stimulation time is about 48 hours or at least about 48 hours.

[0121] Other conditions for stimulating or activating immune cell populations described herein can further include CO2 levels. CO2 levels for stimulation are about 1.0-10% CO2, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2, about 3-7% CO2, about 4-6% CO2, or about 4.5-5.5% CO2. In one embodiment, the CO2 level for stimulation is about 5% CO2.

[0122] Conditions for stimulating or activating an immune cell population may further include temperature, a period of stimulation, and / or the presence of a certain level of CO2, in any combination. For example, stimulating a population of immune cells may include stimulating the population of immune cells with one or more immune cell stimulators at a temperature of about 36-38°C for about 44-52 hours in the presence of a CO2 level of about 4.5-5.5%. One or more immune cells of the present application may be administered to a subject for use in immunotherapy or cell therapy. Thus, one or more immune cells may be harvested from a subject in need of immunotherapy or cell therapy. Once harvested, the one or more immune cells may be treated for any suitable period of time before being administered to a subject.

[0123] The concentration, quantity, or population of lymphocytes or resulting products produced by the methods herein may be about 1.0-10.0 x 10 6 In a specific embodiment, the concentration is about 1.0 to 2.0 x 10 cells / mL. 6 cells / mL, approximately 1.0~3.0×10 6 cells / mL, approximately 1.0~4.0×10 6 cells / mL, approximately 1.0~5.0×10 6 cells / mL, approximately 1.0~6.0×10 6 cells / mL, approximately 1.0~7.0×10 6 cells / mL, approximately 1.0~8.0×10 6 cells / mL, 1.0~9.0×10 6 cells / mL, approximately 1.0~10.0×10 6 cells / mL, approximately 1.0~1.2×10 6 cells / mL, approximately 1.0~1.4×106 cells / mL, approximately 1.0~1.6×10 6 cells / mL, approximately 1.0~1.8×10 6 cells / mL, approximately 1.0~2.0×10 6 cells / mL, at least approximately 1.0 x 10 6 cells / mL, at least approximately 1.1 x 10 6 cells / mL, at least approximately 1.2 x 10 6 cells / mL, at least approximately 1.3 x 10 6 cells / mL, at least approximately 1.4 x 10 6 cells / mL, at least approximately 1.5 x 10 6 cells / mL, at least approximately 1.6 x 10 6 cells / mL, at least approximately 1.7 x 10 6 cells / mL, at least approximately 1.8 x 10 6 cells / mL, at least approximately 1.9 x 10 6 cells / mL, at least approximately 2.0 x 10 6 cells / mL, at least approximately 4.0 x 10 6 cells / mL, at least approximately 6.0 × 10 6 cells / mL, at least approximately 8.0 × 10 6 cells / mL, or at least about 10.0 x 10 6 cells / mL.

[0124] An anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of anti-CD3 and anti-CD28 antibodies can be used in conjunction with or independently of exposing one or more cells obtained from a donor subject to hypoxic culture conditions in the presence or absence of pressure above atmospheric pressure to stimulate a lymphocyte population. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibody or 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 commercially purchased from suppliers known in the art, including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Additionally, one of skill in the art would understand how to generate anti-CD3 and / or anti-CD28 antibodies by standard methods. In some aspects, the one or more T cell stimulatory agents used in accordance with the step of stimulating a population of lymphocytes comprise an antibody or functional fragment thereof that targets a T cell stimulatory or costimulatory molecule in the presence of a T cell cytokine. In one embodiment, the one or more T cell stimulatory agents comprise an anti-CD3 antibody and IL-2. In a specific embodiment, the T cell stimulatory agent comprises an anti-CD3 antibody at a concentration of 50 ng / mL. The concentration of anti-CD3 antibody is about 20 ng / mL to 100 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In alternative embodiments, T cell activation is not required.

[0125] The methods described herein further include transducing the population of activated immune cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using one or more cycles of viral transduction to generate a population of transduced immune cells. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. Viral vectors that can be used in accordance with the transduction step can be any ecotropic or amphotropic viral vector, including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors. The method further includes transducing one or more immune cells with a retrovirus. In one aspect, the viral vector used to transduce the population of activated immune cells is an MSGV1γ retroviral vector. In one embodiment, the viral vector used to transduce the population of activated immune cells is the PG13-CD19-H3 vector described in Kochenderfer, J. Immunother. 32(7):689-702 (2009). According to one aspect of this embodiment, the viral vector is propagated in suspension culture in a medium specific for viral vector production, referred to herein as the viral vector inoculum. Any suitable growth medium and / or supplements for growing viral vectors can be used in the viral vector inoculum according to the methods described herein. According to some aspects, the viral vector inoculum is then added to the serum-free culture medium described below during the transduction step. In some aspects, 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., a tumor cell. Optionally, in addition to exposing one or more cells obtained from a donor subject to hypoxic culture conditions in the presence or absence of pressure above atmospheric pressure, the conditions for transducing a population of activated immune cells described herein can include a specific time at a specific temperature and / or in the presence of a specific level of CO2.The temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34 to 38°C, about 35 to 37°C, about 36 to 38°C, or about 36 to 37°C. In one embodiment, the temperature for transduction is about 37°C. The predetermined temperature for transduction may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C, about 34 to 39°C, or about 35 to 37°C. In one embodiment, the predetermined temperature for transduction may be about 36 to 38°C, about 36 to 37°C, or about 37°C. The time for transduction is about 12 to 36 hours, about 12 to 16 hours, about 12 to 20 hours, about 12 to 24 hours, about 12 to 28 hours, about 12 to 32 hours, about 20 hours, or at least about 20 hours, and is about 16 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 for transduction is about 1.0 to 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 to 7% CO2, about 4 to 6% CO2, about 4.5 to 5.5% CO2, or about 5% CO2.

[0126] Introducing a population of activated immune cells as described herein can be performed in any combination for a period of time, at a particular temperature, and / or in the presence of a particular level of CO2, such as at a temperature of about 36-38°C for about 16-24 hours, and in the presence of a CO2 level of about 4.5-5.5% CO2. The immune cells can be prepared by combining any one of the methods of the present application with any manufacturing method for preparing T cells for immunotherapy, including, but not limited to, those described in International Publication Nos. 2015 / 120096 and 2017 / 070395, which are incorporated herein by reference for purposes of describing these methods, any and all methods used to prepare axicabtagenecilloreucel or Yescarta®, any and all methods used to prepare tisagenlecleucel / Kymriah™, any and all methods used to prepare "off-the-shelf" T cells for immunotherapy, and any other method of preparing lymphocytes for administration to humans. The manufacturing process can be adapted to remove circulating tumor cells from cells obtained from a patient.

[0127] The CAR-T cells may be engineered to express other molecules and may be any one of the following exemplary types or others available in the art: first, second, third, fourth, fifth, or higher generation CAR-T cells, armored CAR-T cells, motile CAR-T cells, TRUCK T cells, switch receptor CAR-T cells, gene-edited CAR T cells, dual receptor CAR T cells, suicide CAR T cells, drug-inducible CAR-T cells, synNotch-inducible CAR T cells, and inhibitory CAR T cells. In one aspect, the T cells are autologous T cells. In one aspect, the T cells are autologous stem cells (for autologous stem cell therapy or ASCT). In one aspect, the T cells are non-autologous T cells.

[0128] Cells (such as immune cells or T cells) are either genetically modified after isolation or selection using known methods, or activated and / or expanded in vitro (or differentiated in the case of progenitor cells) before being genetically modified. Immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) as described herein and activated and / or expanded in vitro. Methods for activating and expanding T cells can be found in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, as well as WO 2012 / 079000, the entire contents of which are incorporated herein by reference. Generally, such methods may include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, e.g., anti-CD3 and / or anti-CD28 antibodies, which may be bound to beads or other surfaces, in a culture medium containing specific cytokines, such as IL-2. The Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells, can be used. T cells can be activated and stimulated and expanded with suitable support cells, antibodies, and / or cytokines as described in U.S. Patent Nos. 6,040,177 and 5,827,642 and WO 2012 / 129514, the contents of which are incorporated herein by reference in their entirety.

[0129] The cell surface receptor expressed by the engineered immune cells can be any antigen or molecule targeted by the CAR, such as an anti-CD19 CAR, FMC63-28Z CAR, or FMC63-CD828BBZ CAR (Kochenderfer et al., J Immunother. 2009, 32(7):689; Locke et al., Blood 2010, 116(20):4099, the subject matter of both of which is incorporated herein by reference). In certain aspects, the predetermined dose of engineered immune cells can be greater than about 1 million to less than about 3 million transduced T cells / kg. In one embodiment, the predetermined dose of engineered T cells can be about 1 million to about 2 million transduced T cells per kilogram of body weight (cells / kg). A predetermined dose of engineered T cells can be from 1 million to about 2 million, at least about 2 million to less than about 3 million transduced T cells per kilogram of body weight (cells / kg). In one embodiment, a predetermined dose of engineered T cells can be about 2 million transduced T cells / kg. In another embodiment, a predetermined dose of engineered T cells can be at least about 2 million transduced 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 modified T cells / kg.

[0130] The methods described herein involve expanding or enriching a population of one or more transduced immune cells for a period of time to produce a population of engineered immune cells. The period of expansion can be any suitable period that allows for the production of (i) a sufficient number of cells in the population of engineered immune cells for at least one dose to be administered to a patient, (ii) a population of engineered immune cells with a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This period of time depends on the cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and other variables. The predetermined period of expansion 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. In one embodiment, the period of expansion in the present method is shortened compared to methods known in the art. For example, the period of a given expansion may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. In one example, the period of expansion is about 3 days and the time from enrichment of the lymphocyte population to generation of the engineered immune cells is about 6 days.

[0131] Conditions for expanding the population of transduced immune cells can include temperature and / or the presence of a certain level of CO. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 35-37°C, about 36-37°C, or about 37°C. The CO level is 1.0-10% CO, 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% CO, about 4.5-5.5% CO, about 5% CO, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO.

[0132] Each step of the method described herein can be carried out in a closed system. The closed system can be a closed-bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife Cell Culture bags). The cell culture bag used in the closed-bag culture system can be coated with a recombinant human fibronectin fragment during the transduction step. The recombinant human fibronectin fragment can contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can be used to increase the gene efficiency of retroviral transduction of immune cells by assisting in colocalization of the target cell with the viral vector. In one embodiment, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). The cell culture bag may contain about 1 to 60 μg / mL, about 1 to 40 μg / mL, about 1 to 20 μg / mL, 20 to 40 μg / mL, 40 to 60 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, about 20 μg / mL, about 2 to 5 μg / mL, about 2 to 10 μg / mL, about 2 to 20 μg / mL The surface is coated with the recombinant human fibronectin fragment at a concentration of about 2 to 25 μg / mL, about 2 to 30 μg / mL, about 2 to 35 μg / mL, about 2 to 40 μg / mL, about 2 to 50 μg / mL, about 2 to 60 μg / mL, at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL.In one embodiment, the cell culture bag is coated with at least about 10 μg / mL of recombinant human fibronectin fragment. The cell culture bag used in the closed-bag culture system can optionally be blocked with human serum albumin (HSA) during the transduction process. In another embodiment, the cell culture bag is not blocked with HSA during the transduction process.

[0133] The engineered immune cell population produced by the above method can optionally be cryopreserved to allow for later use of the cells. Methods for cryopreserving engineered immune cell populations are also provided herein. Such methods can include washing and concentrating the engineered immune cell population with a diluent. For example, the diluent can be saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. HSA can also be added to the washed and concentrated cells to improve cell viability and recovery after thawing. In another embodiment, the washing solution is saline, and the washed and concentrated cells are supplemented with HSA (5%). The method can also include generating a cryopreservation mixture, wherein the cryopreservation mixture comprises the diluted cell population in the diluent and a suitable cryopreservation solution. The cryopreservative solution can be any suitable cryopreservative solution, including, but not limited to, CryoStor 10 (BioLife Solutions), and is mixed with the engineered immune cell dilution at a 1:1 or 2:1 ratio. 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 cryopreserved mixture. Cryopreservation of the engineered immune cell population can include washing the cells with 0.9% saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStor™ CS10 (final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method also includes freezing the cryopreservation mixture. The cryopreservation mixture can also include freezing the engineered immune cell population at a final concentration of about 1 x 10 HSA in the cryopreservation mixture. 6 ~Approx. 1.5×10 7The cells are frozen in a controlled-rate freezer using a defined freezing cycle at a cell concentration of 1000 cells / mL. The method may also include storing the cryopreservation mixture in vapor-phase liquid nitrogen.

[0134] The population of engineered immune cells produced by the methods described herein can be cryopreserved in predetermined doses. The predetermined dose can be a therapeutically effective dose, which can be any of the therapeutically effective doses 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 cell, the nature of the disease or condition being treated, or a combination of both.

[0135] In one embodiment, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered T cells / kg. In certain embodiments, the population of engineered T cells may 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, or at least about 10 million engineered T cells / kg. In other embodiments, the population of engineered T cells may be cryopreserved at 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 certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered T cells / kg. The population of engineered T cells can be cryopreserved at a predetermined volume of about 1 million to about 2 million cells / kg, about 1 million to about 3 million cells / kg, about 1 million to about 4 million cells / kg, about 1 million to about 5 million cells / kg, about 1 million to about 6 million cells / kg, about 1 million to about 7 million cells / kg, about 1 million to about 8 million cells / kg, about 1 million to about 9 million cells / kg, or about 1 million to about 10 million cells / kg. The predetermined dose of the population of engineered T cells can be calculated based on the subject's body weight. In one example, the population of engineered T cells can be cryopreserved in about 0.5 to 200 mL of cryopreservation medium.Furthermore, the population of engineered T cells may be cryopreserved in about 0.5 mL, about 1.0 mL, about 5.0 mL, about 10.0 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL, about 10-30 mL, about 10-50 mL, about 10-70 mL, about 10-90 mL, about 50-70 mL, about 50-90 mL, about 50-110 mL, about 50-150 mL, or about 100-200 mL of cryopreservation medium. In certain embodiments, the population of engineered T cells may be cryopreserved in preferably about 50-70 mL of cryopreservation medium.

[0136] In one embodiment, at least one of (a) contacting the population of immune cells with exogenous IL-2, exogenous IL-7, exogenous IL-15, and / or other cytokines, (b) stimulating the population of immune cells, (c) transducing the activated population of immune cells, and (d) expanding the transduced population of immune cells is performed using a serum-free culture medium that does not contain added serum. In some aspects, each of (a) through (d) is performed using a serum-free culture medium that does not contain added serum. As referred to herein, the term "serum-free medium" or "serum-free culture 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 medium as a separate, distinct component for the purpose of supporting the viability, activation, and growth of the cultured cells. Any suitable immune cell growth medium can be used to culture cells in suspension according to the methods described herein. For example, immune cell growth medium can include, but is not limited to, a sterile low-glucose solution containing suitable amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OPTMIZER™ (Life Technologies). In contrast to typical methods for generating engineered immune cells, the methods described herein can use culture medium that is not supplemented with serum (e.g., human or bovine).

[0137] The present application provides various methods of treating cancer using T cells. In one embodiment, the T cells are CD19-directed CAR-T cells and can be prepared by combining any one of the methods of the present application with any step of a manufacturing method for preparing T cells for immunotherapy, including, but not limited to, those described in International Publication Nos. 2015 / 120096 and 2017 / 070395, both of which are incorporated herein by reference for purposes of describing these methods, any and all methods used to prepare axicabtageneciloleucel or Yescarta®, any and all methods used to prepare tisagenlecleucel / Kymriah™, any and all methods used to prepare "off-the-shelf" T cells for immunotherapy, and any other method for preparing lymphocytes for administration to humans. In some embodiments, the manufacturing process is adapted to specifically remove circulating tumor cells from cells obtained from a patient.

[0138] In one aspect, the T cells are CD19 CAR-T cells prepared by the methods described in International Application No. US2016 / 057983. In one embodiment, a population of circulating tumor cell-depleted T cells is prepared from a leukapheresis product. These cells may be prepared as described in International Application No. US2016 / 057983 and are further described herein as CD19 CAR-T cells. Briefly, CD19 CAR-T is an autologous CAR T cell product in which a 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 CD19-expressing cells. CD19 +After CAR engagement with target cells, 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 costimulatory signals that function with the primary CD3ζ signal to enhance T cell function, including interleukin (IL)-2 production. Together, these signals can stimulate CAR T cell proliferation and induce target cell killing. Furthermore, 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 CD19 CAR-T cells can include FMC63-28Z.

[0139] Due to the presence of circulating tumor cells in certain cancers, the manufacturing of CD19 CAR-T is expected to be more efficient than CD4 + and CD8 + Include a T cell enrichment step. The T cell enrichment or isolation step can reduce circulating CD19-expressing tumor cells in the leukapheresis material and can be associated with the activation, expansion, and depletion of anti-CD19 CAR T cells during manufacturing.

[0140] The methods described herein can improve the therapeutic outcome or efficacy of immune or cellular therapy, which may 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 method of selection, in vitro enrichment, and administration of a patient's autologous or allogeneic T cells capable of recognizing and binding to tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, enriched in vitro, and administered to a patient. TIL cells can be either autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that involves isolating T cells capable of targeting tumor cells from a patient, enriching the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T cell transplantation can include transplantation of ex vivo expanded naturally occurring T cells or genetically engineered T cells. As explained in more detail above, engineered autologous cell therapy is adoptive T cell therapy, in which a patient's own lymphocytes are isolated, genetically modified to express tumor-targeting molecules, expanded in vitro, and administered back to the patient. Non-T cell transplants can include autologous or allogeneic therapy using non-T cells, such as, but not limited to, natural killer (NK) cells.

[0141] The immune cell therapy of the present application is an engineered autologous cell therapy (eACT™). According to this aspect, the method can include harvesting immune cells from a donor. The isolated immune cells can then be contacted with an exogenous activating reagent (e.g., a cytokine), expanded, 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, one or more immune cells are transduced with a retrovirus containing 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., 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 can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor can include a binding molecule against a tumor antigen. The binding molecule can 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, as well as any fragment or combination thereof. The chimeric antigen receptor may further comprise a hinge region. The hinge region may be derived from that of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α. In one embodiment, the hinge region is derived from that of IgG4. The chimeric antigen receptor may also comprise a transmembrane domain. The transmembrane domain may be that of any transmembrane molecule that is a co-receptor on an immune cell or a member of the immunoglobulin superfamily. In a specific embodiment, the transmembrane domain is derived from that of CD28, CD28T, CD8α, CD4, or CD19. In another embodiment, the transmembrane domain comprises a domain derived from the CD28 transmembrane domain. In another embodiment, the transmembrane domain comprises a domain derived from the CD28T transmembrane domain. The chimeric antigen receptor may further comprise one or more costimulatory signaling regions.For example, the costimulatory signaling region can be the signaling region of CD28, CD28T, OX-40, 41BB, CD27, inducible T cell costimulatory molecule (ICOS), CD3γ, CD3δ, CD3ε, CD247, Igα (CD79a), or an Fcγ receptor. In a further 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.

[0142] In some embodiments, the tumor antigen is 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 / mutant), BCMA (B cell maturation antigen), Bcr-abl (cleavage prone 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 40), CD44v9 (cluster of differentiation 50), CD44v10 (cluster of differentiation 60), CD44v11 (cluster of differentiation 70), CD44v12 (cluster of differentiation 80), CD44v13 (cluster of differentiation 90), CD44v14 (cluster of differentiation 150), CD44v15 (cluster of differentiation 160), CD44v16 (cluster of differentiation 170), CD44v17 (cluster of differentiation 180), CD44v18 (cluster of differentiation 190), CD44v19 (cluster of differentiation 190), CD44v19 (cluster of differentiation 190), CD44v19 (cluster of differentiation 190), CD44v16 (cluster of differentiation 190), CD44v19 ... Class of Cellular Expression 44, exon 7 / 8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27, mutant), CDK4 / m (cyclin-dependent kinase 4, mutant), CEA (carcinoembryonic antigen), CT (cancer / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen, melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein Protein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2, mutant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100 kDa), HAGE (helix course antigen), HER-2 / neu (human epithelial receptor-2 / neuronal; also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2, mutated), HST-2 (human signet ring tumor-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-1 recognized by T cells / melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin / m (myosin, mutant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous, mutant 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 minor bcr-abl (190KD bcr-abl protein), 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 ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor rejection squamous antigen 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 (triosephosphate isomerase, mutated), 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.

[0143] T cell therapy involves administering engineered T cells ("engineered TCR T cells") to a patient that express a T cell receptor. The T cell receptor (TCR) can include a molecule that binds to a tumor antigen. In some embodiments, the tumor antigen is 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2 / neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIAA0205, KDR, κ-light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, 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 Selected from the group consisting of minor bcr-abl, Pml / RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF-R2, WT1, and any combination thereof.

[0144] "CD19-directed genetically modified autologous T cell immunotherapy" refers to a suspension of chimeric antigen receptor (CAR)-positive immune cells. An example of such immunotherapy is Clear CAR-T therapy, which uses CAR-T cells that are free of circulating tumor cells and enriched for CD4+ / CD8+ T cells. Another example is axi-cel™ (also known as YESCARTA®). See Kochenderfer, et al., (J Immunother 2009;32:689 702). Other non-limiting examples include JCAR017, JCAR015, JCAR014, Kymriah (tisagenlecleucel), Uppsala U. anti-CD19 CAR (NCT02132624), and UCART19 (Celectis). See Sadelain et al. Nature Rev. Cancer Vol. 3 (2003); Ruella et al., Curr Hematol Malig Rep., Springer, NY (2016); and Sadelain et al. Cancer Discovery (Apr 2013). To prepare for CD19-directed genetically modified autologous T cell immunotherapy, a patient's own T cells can be harvested and genetically modified ex vivo by retroviral transduction to express a chimeric antigen receptor (CAR) comprising a murine anti-CD19 single-chain variable fragment (scFv) linked to CD28 and CD3-ζ costimulatory domains. In some embodiments, the CAR comprises a murine anti-CD19 single-chain variable fragment (scFv) linked to 4-1BB and CD3-ζ costimulatory domains. The anti-CD19 CAR T cells can be expanded and infused back into the patient, where they are capable of recognizing and eliminating CD19-expressing target cells.

[0145] 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 breakpoint 2 (SSX2), melanoma antigen (MAGE), and any combination thereof. In another embodiment, the TCR comprises a binding molecule to a lineage-specific antigen. In a further embodiment, the antigen is selected from the group consisting of melanoma antigen-1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof, recognized by T cells. In certain embodiments, the T cell therapy comprises administering to the patient engineered CAR T cells that express a chimeric antigen receptor that binds to CD19 and further comprise a CD28 costimulatory domain and a CD3-zeta signaling region. In additional embodiments, the T cell therapy comprises administering to the patient KTE-C19.In one embodiment, the antigenic moiety further includes an Epstein-Barr virus (EBV) antigen (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), a hepatitis A virus antigen (e.g., VP1, VP2, VP3), a hepatitis B virus antigen (e.g., HBsAg, HBcAg, HBeAg), a hepatitis C virus antigen (e.g., envelope glycoproteins E1 and E2), a herpes simplex virus type 1, 2, or 8 (HSV1, HSV2, or HSV8) viral antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), a cytomegalovirus (C Examples of antigenic moieties include, but are not limited to, human immunodeficiency virus (HIV) viral antigens (e.g., glycoproteins gB, gC, gE, gG, gH, gI, gJ, gK, gL, gM, or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoproteins gp120, gp41, or p24), influenza viral antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps viral antigens, human papillomavirus (HPV) viral antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) viral antigens, or varicella-zoster virus antigens. In such embodiments, the cell surface receptor can be any TCR or any CAR that recognizes any of the foregoing viral antigens on target virus-infected cells. In other embodiments, the antigenic moiety is associated with a cell with an immune or inflammatory dysfunction. Such antigenic moieties 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 (HSPs), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0146] The methods disclosed herein can include T cell therapy, which involves the transplantation of one or more T cells into a patient. The T cells can 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, 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, a therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In one embodiment, a therapeutically effective amount of CAR T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 In one embodiment, the amount of CD19 CAR-T cells is 2×10 6 cells / kg, with a maximum dose of 2 x 10 for subjects ≥ 100 kg 8In another embodiment, the amount of CD19 CAR-T cells is 0.5 x 10 6 cells / kg, with a maximum dose of 0.5 x 10 for subjects ≥ 100 kg 8 It is a cell.

[0147] The patient may be preconditioned or lymphodepleted prior to 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 chemotherapeutic agents and / or radiation therapy. In some embodiments, preconditioning may include any treatment that reduces the number of endogenous lymphocytes, eliminates cytokine sinks, increases serum levels of one or more homeostatic cytokines or proinflammatory factors, enhances effector function of T cells administered after treatment, enhances activation and / or availability of antigen-presenting cells, or any combination thereof prior to T cell therapy. Preconditioning may include increasing serum levels of one or more cytokines in the subject. The method may further include administering a chemotherapeutic agent. The chemotherapeutic agent may be a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens, along with correlating beneficial biomarkers, are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety. These include, for example, methods of pretreating patients in need of T cell therapy, including administering a designated beneficial dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day) and the specified dose of fludarabine (20 mg / m 2 / day~900mg / m 2 / day) to the patient. One such dosing regimen includes administering about 500 mg / m to the patient prior to administering a therapeutically effective amount of engineered T cells to the patient. 2 / day cyclophosphamide, and approximately 60 mg / m 2 In one embodiment, the conditioning regimen comprises administering fludarabine at 500 mg / m² for three days.2 + Fludarabine 30 mg / m 2 They may be administered on days -4, -3, and -2, or on days -5, -4, and -3 (day 0 is the day of administration of the cells). In one embodiment, the conditioning regimen includes cyclophosphamide 200 mg / m 2 , 250 mg / m 2 , 300 mg / m 2 , 400v, 500mg / m 2 fludarabine 20 mg / m daily for 2, 3, or 4 days 2 , 25 mg / m 2 , or 30 mg / m 2 In one embodiment, leukapheresis is followed by conditioning chemotherapy (fludarabine 30 mg / m 2 / day and cyclophosphamide 500 mg / m 2 / day) on days -5, -4, and -3, followed by an intravenous infusion of a suspension of CD19 CAR-T cells. In some embodiments, the intravenous infusion time is 15-120 minutes. In one embodiment, the intravenous infusion time is 1-240 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes. In some embodiments, the intravenous infusion time is up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 minutes. In some embodiments, the infusion volume is 50-100 mL. In some embodiments, the infusion volume is 20-100 mL. In some embodiments, the infusion volume is about 30, 35, 40, 45, 50, 55, 60, or about 65 mL. In some embodiments, the infusion volume is about 68 mL. In some embodiments, the suspension is frozen and used within 6, 5, 4, 3, 2, or 1 hour of thawing. In some embodiments, the suspension is not frozen. In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is agitated during infusion. In some embodiments, the immunotherapy is administered within 3 hours of thawing. In some embodiments, the suspension further comprises albumin. In some embodiments, the albumin is present in an amount of about 2-3% by volume. In some embodiments, the albumin is present in an amount of about 2.5% by volume. In some embodiments, the albumin is present in an amount of about 1%, 2%, 3%, 4%, or 5% (v / v). In some embodiments, the albumin is human albumin. In some embodiments, the suspension further comprises DMSO. In some embodiments, the DMSO is present in an amount of about 4-6% by volume. In some embodiments, the DMSO is present in an amount of about 5% by volume. In some embodiments, DMSO is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v / v).

[0148] The methods disclosed herein can be used to treat cancer in a subject, to reduce tumor size, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to eliminate tumors from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to 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.

[0149] Cancers that can be treated include non-vascularized tumors, tumors that are not yet substantially vascularized, or vascularized tumors. Cancers can also include solid or non-solid tumors.

[0150] In one embodiment, the method can be used to treat B-cell malignancies with high levels of circulating CD19-expressing tumor cells, and would be indicated for a patient population characterized by high unmet need.

[0151] Example Treatments for MCL

[0152] In some embodiments, the malignant tumor can be mantle cell lymphoma (MCL). MCL is an aggressive subtype of non-Hodgkin's lymphoma (NHL). MCL accounts for approximately 6% of all new cases of NHL in the United States (US) and 5% to 7% of malignant lymphomas in Western Europe. The estimated annual incidence of MCL is approximately 1 to 2 per 100,000 people in the US and Europe. MCL is more likely to affect men than women, and the median age at diagnosis is 68 years. In some embodiments, r / r MCL is r / r for treatment with allogeneic stem cell transplantation (allo-SCT), which can produce long-term remissions in approximately 25% of patients with relapsed or refractory durable (r / r) MCL if the disease has been shown to be chemotherapeutic-sensitive prior to transplantation, but is also associated with a treatment-related mortality rate of up to 40%.

[0153] In some embodiments, r / r MCL is refractory to treatment with bortezomib, lenalidomide, and temsirolimus, which by themselves result in ORRs ranging from 22% to 32%. Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib and acalabrutinib, result in ORRs of 68% and 81%, respectively, in patients with r / r MCL. However, most patients progress after BTK inhibitor treatment, and outcomes with salvage therapy are poor, with ORRs ranging from 20% to 42%, median duration of response (DOR) of 3 to 5.4 months, and median OS of 2.5 to 9 months. In some embodiments, the present disclosure provides that intervention with CAR T cells can be used to treat cancers with a high Ki67 tumor proliferation index (≥ 30% or ≥ 50%) and poor prognostic factors, such as mutated TP53. In some embodiments, the cancer is MCL. In some embodiments, the morphology of MCL is classical, pleomorphic, or blastoid. In some embodiments, the Ki-67 index can be between 5% and 80%. In some embodiments, the Ki-67 index is about 38%. In some embodiments, high-risk patients have a Ki-67 of ≥ 50% and / or a TP53 mutation by next-generation sequencing. In some embodiments, the patient is 18 years of age or older. In some embodiments, MCL is pathologically confirmed by evidence of either cyclin D1 overexpression and / or the presence of t(11:14).

[0154] In some embodiments, CAR T cell intervention involves depleting circulating lymphoma cells, enriching for CD4+ / CD8+ T cells by positive selection of mononuclear cells from a leukapheresis sample activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transducing T cells from the T cell population with a replication-deficient viral vector containing an anti-CD19 CAR construct. In some embodiments, the CAR construct is FMC63-28Z CAR. CAR T cells generated using this method may be referred to as KTE-X19. In some embodiments, the cells are autologous. In some embodiments, the cells are xenogeneic. In some embodiments, the dose of CAR-positive T cells is 2×106 In some embodiments, the dose of CAR-positive T cells is 1 x 10 anti-CD19 CAR T cells / kg. 6 anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1.6 x 10 6 Anti-CD19 CAR T cells / kg, 1.8×10 6 anti-CD19 CAR T cells / kg, or 1.9 × 10 6 anti-CD19 CAR T cells / kg. In some embodiments, the CD19 CAR construct comprises a CD3ζ T cell activation domain and a CD28 signaling domain.

[0155] In some embodiments, CAR T cells are administered at 25 mg / m on days −5, −4, and −3 after leukapheresis. 2 / day fludarabine and 900 mg / m on day -2 2 / day cyclophosphamide pretreatment is administered as a single infusion on day 0. In some embodiments, the pretreatment is 300 mg / m 2 / day cyclophosphamide, and 30 mg / m for 3 days 2 In some embodiments, the conditioning chemotherapy comprises 30 mg / m fludarabine. 2 / day fludarabine and 500 mg / m on days -5, -4, and -3 2 100 mg / day cyclophosphamide. In some embodiments, the patient may also receive acetaminophen and diphenhydramine or another H1 antihistamine about 30-60 minutes prior to the anti-CD19 CAR T cell infusion. In some embodiments, the patient receives one or more additional doses of anti-CD19 CAR T cells.

[0156] In some embodiments, the MCL cancer is relapsed / refractory MCL (r / r MCL). In some embodiments, the patient has received one or more prior therapies. In some embodiments, the patient has received 1 to 5 prior therapies. In some embodiments, the prior therapies may include autologous SCT, anti-CD20 antibodies, anthracycline- or bendamustine-containing chemotherapy, and / or a Bruton's tyrosine kinase inhibitor (BTKi). In some embodiments, the BTKi is ibrutinib (Ibr). In some embodiments, the BTKi is acalabrutinib (Acala). In some embodiments, the present disclosure provides that MCL patients previously treated with ibrutinib had a more pronounced response to anti-CD19 CAR T-cell therapy compared to patients previously treated with acalabrutinib. Thus, the present disclosure provides methods of treating r / r MCL with anti-CD19 CAR T cell therapy, where the patient has previously received ibrutinib or acalabrutinib treatment and the cancer is preferably relapsed / refractory to that treatment. In some embodiments, the BTKi is tirabrutinib (ONO-4059), zanubrutinib (BGB-3111), CGI-1746, or spebrutinib (AVL-292, CC-292).

[0157] In some embodiments, the disclosure provides that for patients who received prior treatment with Ibr, Acala, 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 for anti-CD19 CAR T cell therapy in MCL patients were 94% / 65% in patients who received prior Ibr treatment, 80% / 40% in patients who received prior Acala treatment, and 100% / 100% in patients who received both BTKi treatments. In some embodiments, the 12-month survival rates for patients who received prior Ibr, Acala, or both treatments were 81%, 80%, or 100%, respectively. In some embodiments, CAR T cell expansion is associated with the ORR / CR rates in patients who received prior Ibr and / or Acala treatment. Thus, in one embodiment, a patient is treated with both Ibr and Acala. In one embodiment, the disclosure provides a method for predicting ORR / CR in MCL patients previously treated with Ibr and / or Acala by measuring peak CAR T cell levels and comparing them to a reference standard. In one embodiment, the disclosure provides a method for predicting continued response based on measuring peak CAR T cell levels / baseline tumor burden (CEN and INV). In one embodiment, the higher the ratio, the greater the likelihood of continued response at / by 12 months. In one embodiment, a ratio of 0.00001 to 0.005 predicts non-response at / by 12 months. In one embodiment, a ratio of 0.006 to 0.3 predicts relapse at / by 12 months. In one embodiment, a ratio of 0.4 to 1 predicts continued response at / by 12 months. In one embodiment, the ratio can be determined from an average population by one of skill in the art.

[0158] In some embodiments, additional inclusion criteria include those listed in Example 2. In some embodiments, additional exclusion criteria include those listed in Example 2.

[0159] In some embodiments, patients may have received bridging therapy (post-leukapheresis and pre-chemotherapy) with dexamethasone (e.g., 20-40 mg or equivalent PO or IV daily for 1-4 days), methylprednisolone, ibrutinib (e.g., 560 mg PO daily), and / or acalabrutinib (e.g., 100 mg PO twice daily) after leukapheresis, e.g., completed within 5 days 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 immunomodulatory agent, R-CHOP, bendamustine, an alkylating agent, and / or a platinum-based agent.

[0160] In some embodiments, the present disclosure provides that all MCL patients who responded to CAR T cell infusion achieved T cell proliferation, while no proliferation was observed in non-responding patients. In some embodiments, the response is an objective response rate (complete response + partial response). The present disclosure provides that CAR T cell levels correlate with ORR over the first 28 days, as measured by the area under the curve (AUC) from days 0 to 28. 0-28 ) and peak levels were more than 200-fold higher in responders versus non-responders, which is due to minimal residual disease (MRD, sensitivity 10 -5 This suggests that greater expansion leads to a better and potentially more meaningful response, as indicated by the >80-fold higher Peak / AUC CAR T cell levels in MCL-negative patients compared to MRD-positive patients (week 4). Accordingly, the present disclosure provides methods for predicting patient response and MRD to CAR T-cell therapy for MCL, comprising measuring Peak / AUC CAR T cell levels and comparing them to a reference standard. In some embodiments, peak CAR T-cell expansion is observed 8-15 days after CAR T-cell administration. In some embodiments, CAR T-cell levels are measured by qPCR. In some embodiments, peak CAR T-cell levels, AUC 0-28and / or MRD is monitored by next-generation sequencing. In some examples, CAR T cell number is measured as the number of cells in 1 microliter of blood. In some examples, CAR T cell number is measured by the number of copies of the CAR gene in 1 μg of host DNA. In some examples, CAR T cell number is measured as described in Kochenderfer JN et al. J. Clin. Oncol. 2015;33:540-549. In one embodiment, CAR T cell levels are measured as described in Locke FL et al. Mol Ther. 2017;25(1):285-295.

[0161] In some embodiments, the present disclosure provides that there is a difference in T cell proliferation between responders and non-responders. In some embodiments, the present disclosure provides that the median peak anti-CD19 CAR T cell level in responders (those with complete and partial remission) was 102.4 cells / μL (range: 0.2-2589.5 cells / μL, n=51) and in non-responders was 12.0 cells / μL (range: 0.2-1364.0 cells / μL, n=8). In some embodiments, the present disclosure provides that the AUC (AUC 0-28 ) was 1487.0 cells / μL·day (range: 3.8–2.77 × 10 4 cells / μL·day, n=51) in non-responders and 169.5 cells / μL·day (range: 1.8–1.17 10 × 10 4 cells / μL·day, n=8). Peak (24.7 cells / μL) anti-CD19 CAR T cells (peak: and AUC 0-28 The median levels (360.4 cells / μL·day) were similar to those in patients receiving corticosteroids alone (n=2) (peak: 24.2 cells / μL, AUC 0-28 In patients receiving tocilizumab alone (n=10), the mean peak anti-CD19 CAR T cell count was 86.5 cells / μL, with an AUC0-28 In patients receiving both corticosteroids and tocilizumab (n = 37), the mean peak was 167.2 cells / μL, and the AUC 0-28 The median peak anti-CD19 CAR T cell value was 74.1 cells / μL in patients aged 65 years or older (n=39) and 112.5 cells / μL in patients younger than 65 years (n=28). AUC of anti-CD19 CAR T cells 0-28 The median values ​​were 876.5 cells / μL·day in patients aged 65 years and older and 1640.2 cells / μL·day in patients younger than 65 years. Gender was associated with the AUC of anti-CD19 CAR T cells. 0-28 and C max Thus, the present disclosure provides a method for predicting response in MCL, comprising measuring T cell proliferation after anti-CD19 CART treatment and comparing the levels to a reference standard.

[0162] In some embodiments, the present disclosure provides that CAR T-cell proliferation was higher in MCL patients with grade >= 3 than in those with CRS and NE events of grade <= 3. Accordingly, the present disclosure provides a method of predicting CRS and NE events of grade >= 3, comprising measuring CAR T-cell proliferation after CAR T-cell therapy and comparing the level to a reference value; the higher the CAR T-cell proliferation, the higher the likelihood of CRS and NE events of grade >= 3.

[0163] In some embodiments, cytokine levels are measured by protein or mRNA levels (either). In some embodiments, cytokine levels are measured as described in Locke FL et al. Mol Ther. 2017;25(1):285-295.

[0164] In some embodiments, the present disclosure provides that serum GM-CSF and IL-6 peak levels (reached approximately 8 days after CAR T cell administration) were positively associated with grade > 3 CRS and grade > 3 NE in MCL patients. Accordingly, the present disclosure provides a method of predicting grade > 3 CRS and grade > 3 NE comprising measuring peak levels of GM-CSF and IL-6 after CAR T cell administration and comparing them to reference levels; the higher the peak levels of these cytokines, the higher the likelihood of grade > 3 CRS and NE.

[0165] In some embodiments, the present disclosure provides that in MCL patients, serum ferritin was positively associated with CRS of grade > 3. Accordingly, the present disclosure provides a method of predicting CRS of grade > 3, comprising measuring peak levels of serum ferritin after CAR T cell administration and comparing them to reference levels, wherein the higher the peak level of ferritin, the higher the likelihood of CRS of grade > 3.

[0166] In some embodiments, the present disclosure provides that in MCL patients, serum IL-2 and IFNγ were positively associated with NE of grade > 3. Accordingly, the present disclosure provides a method of predicting CRS of grade > 3, comprising measuring peak levels of serum IL-2 and IFNγ after CAR T cell administration and comparing them to reference levels, wherein the higher the peak levels of IL-2 and IFNγ, the higher the likelihood of NE of grade > 3.

[0167] In some embodiments, the disclosure provides that in MCL patients, cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and vascular cell adhesion molecule (VCAM) were positively associated with grade ≧3 NE. Accordingly, the disclosure provides a method of predicting grade ≧3 CRS, 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 levels of C-reactive protein, ferritin, IL-6, IL-8, and / or vascular cell adhesion molecule (VCAM), the higher the likelihood of grade ≧3 NE. In some embodiments, the one or more adverse events are managed according to Table 13 and / or Table 14.

[0168] In some embodiments, the present disclosure provides that peak serum levels of cytokines positively associated with CRS of grade ≧3 included IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and perforin. In some embodiments, the present disclosure provides that peak serum levels of cytokines associated with NE of grade ≧3 included IL-2, IL-1Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. In some embodiments, the present disclosure provides that cytokines associated with both CRS and NE of grade ≧3 included IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B. In some embodiments, cytokine serum levels peak within 7 days of CAR T cell administration. Thus, the present disclosure provides a method of predicting grade ≧3 CRS after CAR T cell administration, comprising measuring peak serum levels of IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and / or perforin after anti-CD19 CAR T treatment and comparing those levels to a reference standard. Thus, the present disclosure also provides a method of predicting grade ≧3 CRS and grade ≧3 NE in MCL, comprising measuring peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B after anti-CD19 CAR T treatment and comparing those levels to a reference standard.

[0169] In some embodiments, the present disclosure provides that MCL patients with mutant TP53 versus wild-type TP53 tended to have increased peak proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1) cytokine levels. Thus, in some embodiments, the present disclosure provides methods of improving response to CAR T cell therapy in MCL, comprising manipulating proliferative and / or inflammatory cytokine levels after CAR T cell administration.

[0170] In some embodiments, the present disclosure provides that patients who were MRD-negative one month after CAR T cell administration tended to have increased peak levels of IFN-γ and IL-6, and increased IL-2, compared to patients who were MRD-positive one month after administration. Accordingly, the present disclosure provides a method of predicting whether a patient will be MRD-negative in MCL, comprising measuring the peak serum levels of IFN-γ, IL-6, and / or IL-2 after anti-CD19 CAR T treatment and comparing those levels to a reference standard.

[0171] In some embodiments, the disclosure provides that the phenotype of the T cell product varied between types of MCL. In some embodiments, the disclosure provides that in the anti-CD19 CAR T product manufactured, the median (range) CD4+ / CD8+ T cell ratio in patients with classical, blastoid, or pleomorphic MCL was 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. The phenotypes (median [range]) of the product T cells included less differentiated CCR7+ T cells (classical 40.0% [2.6-88.8], blastoid 35.3% [14.3-73.4], pleomorphic 80.8% [57.3-88.8]) and effector and effector memory CCR7- T cells (classical 59.9% [11.1-97.4], blastoid 64.8% [26.6-85.7], pleomorphic 19.2% [11.1-42.7]). In some embodiments, the present disclosure provides that the 12-month overall survival rates in patients with classical, blastoid, or pleomorphic MCL were 86.7%, 67.9%, or 100%, respectively. Thus, the present disclosure provides methods for improving the treatment of classical, blastoid, or pleomorphic MCL by manipulating the phenotype of the T cell product administered to the patient.

[0172] Exemplary Treatments for B-Cell ALL

[0173] B-ALL cells typically express CD19, and CD19-targeting CAR T-cell therapy is a therapeutic approach for relapsed / relapsed B-ALL. Pehlivan KC et al. Curr Hematol Malig Rep. 2018;13(5):396-406. Anti-CD19 CAR T-cell therapy containing CD3ζ and CD28 costimulatory domains, developed at the National Cancer Institute (Kochenderfer JN et al. J Immunother. 2009;32(7):689-702, Kochenderfer JN et al. Blood. 2010;116(19):3875-3886), demonstrated a 70% overall remission rate after a median follow-up of 10 months in a phase 1 study in children and adults aged 30 years or younger with relapsed / relapsed B-ALL. Lee DW et al. Lancet. 2015;385(9967):517-528. A similar CAR construct evaluated in a phase 1 trial in adults with relapsed / relapsed B-ALL resulted in an 83% complete remission (CR) rate and a median OS of 12.9 months at a median follow-up of 29 months. Park JH et al. N Engl J Med. 2018;378(5):449-459. In these trials, CAR T cells were prepared from leukapheresis samples that were not enriched for CD4+ / CD8+ T cells.

[0174] In some embodiments, the present disclosure is directed to a T cell product in which circulating lymphoma cells have been depleted and CD4+ / CD8+ T cells have been enriched by positive selection of mononuclear cells from a leukapheresis sample activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and the T cells are then expanded from the T cell population transduced with a replication-deficient viral vector containing an anti-CD19 CAR construct. In some embodiments, such a T cell product 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 xenogeneic. In some embodiments, the dose of CAR-positive T cells is 2×10 6In some embodiments, the dose of CAR-positive T cells is 1 x 10 anti-CD19 CAR T cells / kg. 6 anti-CD19 CAR T cells / kg. In some embodiments, the dose of CAR-positive T cells is 1.6 x 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 T cell product is KTE-X19. In some embodiments, the CD19 CAR construct comprises 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 present disclosure provides that an anti-CAR T cell product prepared as described in the previous paragraph can be used in B-cell ALL and B-cell NHL. In one embodiment, the T cell product has the product characteristics in Table 23. In some embodiments, the product characteristics can be selected from the percentage of T cells of a particular 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 characteristic is the level of IFNγ production (pg / mL) in co-culture with the anti-CD19 CAR T product cells and target CD19-expressing cancer cells (e.g., Toledo) cells mixed at a 1:1 ratio. In one embodiment, IFNγ can be measured in the cell culture medium after 24 hours of incubation using a qualified ELISA. In some embodiments, one or more of these product characteristics are superior to those of anti-CAR T cells prepared from leukapheresis without CD4+ / CD8+ positive cell enrichment. In some embodiments, the superior product characteristics can be selected from an increased percentage of cells with a naive phenotype (CD45RA+CCR7+), a decreased percentage of cells with a differentiated phenotype (CCR7-), a decreased level of IFNγ-producing cells, or an increased level of CD8+ cells. In some embodiments, the anti-CD19 T cell product is a 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 In some embodiments, the product comprises naive-like T cells (CD45RA+CCR7+). In some embodiments, the product comprises T cells that are CD45RA+CCR7+, meaning T cells including stem-like memory cells. N The T cell product comprises naive-like T cells. In some embodiments, the T cell product is KTE-X19. In some embodiments, the KTE-X19 has IFNγ production of 190 pg / mL or greater. In certain embodiments, the KTE-X19 has ≥ 90% CD3+ cells. In some other embodiments, the percentage of NK cells in the KTE-X19 is 0.1% (range 0.0%-2.8%). In some additional embodiments, the CD3 - The percentage of cellular impurities is 0.5% (range 0.3%-3.9%).

[0175] In some embodiments, the cancer is relapsed / refractory B-cell ALL. In some embodiments, the patient is 21 years of age or younger. In some embodiments, the patient is 21 years of age or younger, weighs ≥ 10 kg, and has B-cell ALL that is refractory to first-line therapy, relapsed within 18 months of initial diagnosis, R / R after two or more lines of systemic therapy, or R / R after allogeneic stem cell transplant at least 100 days prior to enrollment. In one embodiment, the cancer is an indolent lymphoma or leukemia. In one embodiment, the cancer is an aggressive B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), mantle cell lymphoma and its blastoid variant, and many types, subtypes, and variants of B-lymphoblastic lymphoma. DLBCL can be DLBCL NOS, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL leg type, or EBV-positive DLBCL of the elderly. Other lymphomas of large B cells include primary mediastinal (thymic) LBCL, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, ALK-positive LBCL, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, and primary body cavity effusion lymphoma. Other types of lymphoma include B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt's lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin's lymphoma, splenic marginal zone B-cell lymphoma, MALT-type extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, hairy cell leukemia, lymphoplasmacytic lymphoma (Waldenstrom's hypergammaglobulinemia), and primary body cavity effusion lymphoma. The cancer can be at any stage from stage 1 to stage 4.

[0176] ALL is a common childhood malignancy, comprising approximately 80% of childhood leukemias and 25% of all childhood cancers. Approximately 20% of pediatric patients do not achieve long-term remission after initial treatment, and the 5-year OS rate is approximately 55%. Hunger SP, et al. N Engl J Med. 2015;373:1541-1552, Sun W, et al. Leukemia. 2018;32:2316-2325, Rheingold SR, et al. J Clin Oncol. 2019;37(suppl,abstr):10008, and Oskarsson T, et al. Haematologica. 2016;101:68-76. Patients who relapse early after initial treatment or have primary refractory disease, those with R / R disease after stem cell transplant, and those with multiple relapses have poor outcomes. Sun W, et al. Leukemia. 2018;32:2316-2325, Rheingold SR, et al. J Clin Oncol. 2019;37(suppl,abstr):10008, Oskarsson T, et al. Hematologica. 2016;101:68-76, Nguyen K, et al. Leukemia. 2008;22:2142-2150, Crotta A, et al. Curr Med Res Opin. 2018;34:435-440, Schrappe M, et al. N Engl J Med. 2012;366:1371-1381. Patients who relapse within 18 months of initial diagnosis generally have a 5-year OS rate of 21% to 28%. Rheingold SR, et al. J Clin Oncol. 2019;37(suppl,abstr):10008, Nguyen K, et al. Leukemia. 2008;22:2142-2150. The likelihood of achieving remission and duration of EFS each decrease with the type of subsequent salvage therapy. Sun W, et al. Leukemia. 2018;32:2316-2325. Outcomes remain poor in children and adolescents with R / R ALL after treatment with the novel therapies blinatumomab and inotuzumab ozogamicin, with 1-year OS rates of approximately 36%, highlighting the need for more effective treatment options.von Stackelberg A,et al.J Clin Oncol.2016;34:4381-4389.10, Bhojwani D,et al.Leukemia.2019;33:884-892.

[0177] In some embodiments, the cancer is B-cell NHL, and key enrollment criteria include age under 18 years, weight ≥ 10 kg, and one or more histologically confirmed measurable lesions; diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS); primary mediastinal large B-cell lymphoma; Burkitt lymphoma (BL); Burkitt-like lymphoma; or B-cell lymphoma intermediate between DLBCL and BL, unspecified. In one embodiment, for NHL treatment, disease may be refractory to first-line therapy, in remission / remission after two or more systemic therapies, or in remission / remission after autologous or allogeneic stem cell transplantation ≥ 100 days prior to enrollment. Patients with acute graft-versus-host disease or chronic graft-versus-host disease requiring treatment within 4 weeks of enrollment are ineligible.

[0178] In some embodiments, these B-cell ALL and / or B-cell NHL patients receive fludarabine 25 mg / m on days −4, −3, and −2. 2 / day, and cyclophosphamide 900 mg / m on day -2 2 After conditioning chemotherapy on day 0, CD4+ / CD8+ enriched anti-CD19 CAR T cells (prepared as described immediately above) were administered at 1 × 10 6 A single injection will be administered at a target dose of anti-CD19 CAR T cells / kg.

[0179] In some embodiments, the disclosure provides for the use of CD4+ / CD8+-enriched / cancer cell-depleted anti-CD19 CAR T cells for the successful treatment of B-cell ALL, where patients are 18 years of age or older and have R / RB-cell ALL defined as resistance to first-line therapy (i.e., first-line refractory), relapse within 12 months of first remission, relapse or resistance after two or more prior systemic therapies, or relapse after allogeneic stem cell transplant (SCT). In some embodiments, patients were required to have ≥5% bone marrow blasts, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate renal, hepatic, and cardiac function. For patients treated with prior blinatumomab, leukemic blasts with CD19 expression ≥90% were required. Patients were required to have Philadelphia chromosome-positive (Ph+) disease, extramedullary complications, and central nervous system (CNS)-2 disease (blasts in the cerebrospinal fluid [CSF], <5 white blood cells / mm3) without neurological changes. 3 ), and Down syndrome were eligible. CNS-3 disease unrelated to neurological changes (blasts in CSF, ≥ 5 leukocytes / mm 3 ) and a history of CNS disorders were excluded. In some embodiments, additional inclusion and exclusion criteria are described in Example 9.

[0180] In some embodiments, the patient may have cancer that is resistant to first-line therapy. In some embodiments, the patient may have cancer that has relapsed after SCT. In some embodiments, the patient may have received prior treatment with blinatumomab, which may be the most recent therapy used prior to anti-CD19 CAR T-cell therapy. In some embodiments, the patient baseline characteristics are any one of the patients listed in Table 18.

[0181] In some embodiments, these B-cell ALL patients receive 2×10 6 , 1×10 6 , or 0.5 × 10 6 In some embodiments, 0.5 x 10 CAR T cells / kg are administered. 6In another embodiment, 0.5 x 10 CAR T cells / kg are administered in a formulation with a total volume of 40 mL. 6 CAR T cells / kg are administered in a formulation with a total volume of 68 mL. In some embodiments, the CAR T cell product is formulated in a total volume of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900, or 1000 mL. In some embodiments, the 40 mL formulation is intended to maintain cell density and cell viability during the freeze / thaw process. In some embodiments, the treatment is related to an adverse event. In some embodiments, one or more adverse events are managed according to any one of Tables 13, 14, 16, or a combination thereof. In some embodiments, one or more adverse events are managed according to the original management guidelines in Table 16. In some embodiments, one or more adverse events are managed according to the revised management guidelines in Table 16. In some embodiments, a vasopressor may be administered to treat CRS. In some embodiments, signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension. In some embodiments, signs or symptoms associated with a neurological event include encephalopathy, convulsions, altered level of consciousness, speech disturbances, tremors, and confusion.

[0182] In some embodiments, patients may have a high disease burden at baseline, as determined by institutional assessment (>25% leukemic blasts in the bone marrow or ≥1,000 blasts / mm in the peripheral circulation). 3 In some embodiments, patients may receive bridging chemotherapy after leukapheresis and before conditioning chemotherapy. In some embodiments, the bridging chemotherapy follows one of the predetermined bridging chemotherapy regimens in Table 17.

[0183] In some embodiments, the conditioning chemotherapy / lymphocyte depletion regimen is administered ≥ 7 days or 5 x half-lives (if shorter) washout from the bridging chemotherapy. In some embodiments, the conditioning chemotherapy / lymphocyte depletion regimen consists of fludarabine intravenously (IV) 25 mg / m on days -4, -3, and -2. 2 / day, and cyclophosphamide IV 900 mg / m on day -2 2 / day. A single infusion of anti-CD19 CAR T cells can be administered on day 0. In some embodiments, a booster infusion of anti-CD19 CAR T cells can be administered thereafter. In some embodiments, patients who achieve a complete response to the first infusion can receive a second infusion of anti-CD19 CAR T cells if they progress after more than 3 months of remission, provided that CD19 expression is maintained and neutralizing antibodies to the CAR are not suspected.

[0184] In some embodiments, droplet digital polymerase chain reaction can be used to measure the presence, proliferation, and persistence of transduced anti-CD19 CAR T cells in the blood. In some embodiments, the procedure is described in Locke FLet et al. Mol Ther. 2017;25(1):285-295. In some embodiments, the present disclosure provides methods of treatment in which the CAR T cell levels are as described in Table 22. In some embodiments, the present disclosure provides that CAR T cells can be undetectable at relapse. The median peak CAR T cell level is 1 x 10 6CAR T cells / kg may be highest and may be similar between patients receiving original and revised AE management. In some embodiments, patients achieving CR / CRi had higher median peak proliferation than non-responders, as did patients with undetectable versus detectable MRD. Higher peak proliferation values ​​were also observed in patients with grade ≥ 3 NE versus 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 per 10,000 viable cells, can be assessed using flow cytometry (NeoGenomics, Fort Myers, FL) according to the methods described in Borowitz MJ, Wood BL, Devidas M, et al. Blood. 2015;126(8):964-971, Bruggemann M. et al. Blood Adv. 2017;1(25):2456-2466, or Gupta S. et al. Leukemia. 2018;32(6):1370-1379.

[0185] In some embodiments, the present disclosure provides that peak levels of several cytokines, chemokines, and pro-inflammatory markers occurred by day 7. In some embodiments, several of these were greater than 2×10 6 In patients receiving CAR T cells / kg, 1 × 10 6In some embodiments, the levels of these proteins / biomarkers tended to be higher (IL-15, CRP, SAA, CXCL10, IFNγ) in those undergoing revised AE management compared to those undergoing original AE management, or lower (IL-6, ferritin, IL-1RA, IFNγ, IL-8, CXCL10, MCP-1) in those undergoing revised AE management compared to those undergoing original AE management. In some embodiments, the levels of these proteins / biomarkers change as described in Figures 9, 10, and 11. Accordingly, in some embodiments, the present disclosure provides methods for using these protein levels as biomarkers for Grade ≥ 3 and / or Grade 0-2 CRS. In some embodiments, the present disclosure provides methods for using these protein levels as biomarkers for Grade ≥ 3 and / or Grade 0-2 CRS according to the values ​​in Figure 11.

[0186] In some embodiments, the present disclosure provides that peak IL-15 serum levels are low in patients with CRS of grade ≧3. In some embodiments, the present disclosure provides that the median peak levels of several pro-inflammatory markers tended to be higher in patients with grade ≧3 CRS and grade ≧3 NE, as described in FIG. 11 (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, Granzyme B. Accordingly, in some embodiments, the present disclosure provides methods for predicting whether a patient will have grade ≧3 CRS by measuring peak levels of serum IL-15 and comparing to a reference standard. In some embodiments, the present disclosure provides methods for predicting whether a patient will have grade ≧3 CRS and / or grade ≧3 NE by measuring peak levels of IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, and / or Granzyme B and comparing to a reference standard. In some embodiments, the present disclosure provides methods for improving anti-CD19 CAR T-cell therapy by administering an agent that reduces the levels of one or more of these biomarkers.

[0187] Reference levels / standards can be established by any method known to those skilled in the art. They serve to identify thresholds or groups of values ​​(e.g., quartiles), from which comparisons can be made to determine which groups, or thresholds above or below which measurements (cytokine levels, CAR T cell counts, etc.) each subject is included in. These groups are established from comparisons of different populations selected as is typical in the art. Depending on where the measurements are included, several treatment characteristics can be predicted, such as objective response, CRS grade, NE grade, etc.

[0188] In certain embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdomyosarcoma-like tumor, central nervous system, 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 (CRL), and / or leukemia-associated cancers. Myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, fibrous histiocytoma malignant type of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, germ cell tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis , Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic islet), Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity 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 cell carcinoma with occult primary midline carcinoma associated with the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplasia syndromes, myelodysplastic / myeloproliferative neoplasms, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma,The cancer may be selected from pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and tumors derived from Wilms' tumor. In certain embodiments, the cancer is treated with KTE-X19.

[0189] In one embodiment, the method may be used to treat a tumor, wherein the tumor is lymphoma or leukemia. Lymphoma and leukemia are cancers of the blood that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell in the bone marrow. This stem cell produces both myeloid and lymphoid progenitor cells, which then give rise to the various types of white blood cells found in the body. White blood cells that arise from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells that arise from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia may affect one or more of these cell types in a patient. In certain embodiments, the tumor is treated with KTE-X19.

[0190] Generally, lymphomas can be divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of cancers originating from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B-cell lymphoma accounts for 80-85% of reported cases. In 2013, it was estimated that there were approximately 69,740 new cases of NHL and more than 19,000 disease-related deaths. Non-Hodgkin's lymphoma is the most common hematologic malignancy and the seventh leading site of new cancers in men and women, accounting for 4% of new cancer cases and 3% of cancer-related deaths. In certain embodiments, lymphoma is treated with KTE-X19.

[0191] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. Approximately 22,000 new cases of DLBCL are diagnosed in the United States each year. It is classified as an aggressive lymphoma, with the majority of patients cured with conventional chemotherapy (NCCN Guidelines for NHL 2014). First-line therapy for DLBCL typically involves an anthracycline-containing regimen containing rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of approximately 80% and a complete response rate of approximately 50%. Approximately one-third of patients are resistant to initial treatment or relapse after R-CHOP. For patients who relapse after responding to first-line therapy, approximately 40–60% can achieve a second response with additional chemotherapy. Standard second-line treatment for autologous stem cell transplant (ASCT)-eligible patients includes rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), with an objective response rate of approximately 63% and a complete response rate of approximately 26%, respectively. Patients who respond to second-line therapy and are deemed sufficiently suitable for transplant undergo consolidation therapy with high-dose chemotherapy and ASCT, which is curative in approximately half of transplant patients. Patients who fail ASCT have a very poor prognosis and no curative treatment options. Primary mediastinal large B-cell lymphoma (PMBCL) has distinct clinical, pathological, and molecular characteristics compared to DLBCL. PMBCL is thought to arise from thymic (medullary) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL typically affects young adults in their 30s, with a slight female predominance. Gene expression profiling suggests deregulated pathways in PMBCL that overlap with Hodgkin lymphoma.Initial therapy for PMBCL generally includes an anthracycline-containing regimen, including rituximab, with or without regional radiation therapy, such as infusion-adjusted etoposide, doxorubicin, and cyclophosphamide with vincristine, prednisone, and rituximab (DA-EPOCH-R). Follicular lymphoma (FL), a B-cell lymphoma, is the most common low-grade (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHL cases. Some patients with FL undergo histological transformation to DLBCL (TFL), a more aggressive form of NHL associated with poorer outcomes. Histological transformation to DLBCL occurs at a rate of approximately 3% per year over a 15-year period, after which the risk of transformation continues to decline. The biological mechanism of histological transformation is unknown. Initial treatment for TFL is influenced by prior treatment for follicular lymphoma and generally includes anthracycline-containing regimens with rituximab to eliminate the aggressive portion of the disease. Treatment options for relapsed / refractory PMBCL and TFL are similar to those for DLBCL. Given the low prevalence of these diseases, no large-scale prospective randomized trials have been conducted in these patient populations. Patients with chemotherapy-resistant disease have a similar or worse prognosis to those with refractory DLBCL. For example, subjects with refractory aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have a high unmet medical need, and further research with new therapies is needed in these populations. In certain embodiments, DLBCL is treated with KTE-X19.

[0192] The CAR T cell therapy of the present disclosure can be administered as a first line therapy, or a second or subsequent line therapy, hi some embodiments, the CAR T cell therapy is administered as a third line therapy, fourth line therapy, fifth line therapy, etc. The selected prior treatment may be any prior anti-cancer therapy, such as, but not limited to, a Bruton's tyrosine kinase inhibitor (BTKi), a checkpoint inhibitor (e.g., anti-PD1 antibody, pembrolizumab (Keytruda), cemiplimab (Libtayo), nivolumab (Opdivo); anti-PD-L1 antibody, atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi); an anti-CTLA-4 antibody, ipilimumab (Yervoy)), an anti-CD19 antibody (e.g., blinatumomab), an anti-CD52 antibody (e.g., alentuzumab); an allogeneic stem cell transplant, an anti-CD20 antibody (e.g., rituximab), systemic chemotherapy, rituximab, an anthracycline, ofatumumab, and combinations thereof. The prior treatment may also be administered in combination with the CD19 CAR T therapy of the present application. In one aspect, eligible patients may have disease refractory to their most recent treatment or may have relapsed within one year after autologous hematopoietic stem cell transplantation (HSCT / ASCT). CAR T-cell therapy may be administered to patients with or suspected of having cancer that is resistant to and / or has relapsed after one or more prior therapies. The cancer may be resistant to first-line therapy (i.e., first-line refractory) or resistant to one or more treatment options. The cancer may have relapsed 12 months after initial remission, relapsed or refractory after two or more prior therapies, or relapsed after HSCT / ASCT. In some embodiments, the cancer is resistant to ibrutinib or acalabrutinib. In some embodiments, the cancer is NHL, and the disease must be first-line refractory, in remission / relapse after two or more systemic therapies, or in remission / relapse after autologous or allogeneic stem cell transplantation 100 days or more prior to enrollment in CAR T-cell therapy and 4 weeks or more off immunosuppressant therapy. In a specific embodiment, the CAR T cell therapy is KTE-X19.

[0193] Thus, the method can be used to treat lymphoma or leukemia, which is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's 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 the group consisting of 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 center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, A LK+ large B-cell lymphoma, plasmablastic lymphoma, primary body cavity effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric 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 The cancer is selected from the group consisting of cutaneous anaplastic large cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sézary 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's lymphoma. In some embodiments, the cancer is resistant to one or more prior therapies and / or the cancer has relapsed after one or more prior therapies.In certain embodiments, the leukemia or lymphoma is treated with KTE-X19.

[0194] In one embodiment, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In other embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is resistant to, or has relapsed after, 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 transplant, or any combination thereof. In one embodiment, the cancer is refractory diffuse large B-cell lymphoma. In certain embodiments, the cancer is treated with KTE-X19.

[0195] 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 lymphoma (BL), Burkitt-like lymphoma, or B-cell lymphoma intermediate between DLBCL and BL, unclassified. In some embodiments, the cancer is relapsed / refractory. In some embodiments, the KTE-X19 therapy is administered as first-line therapy, second-line therapy, or after one or more prior therapies. In some embodiments, the patient is a pediatric patient, an adolescent patient, an adult patient, under 65 years of age, over 65 years of age, or any other age group.

[0196] In some embodiments, compositions comprising immune cells disclosed herein may be administered in combination with any number of additional therapeutic agents. In one embodiment, the additional therapeutic agent is administered simultaneously with T cell therapy. In one embodiment, the additional therapeutic agent is administered before, during, and / or after T cell therapy. In one embodiment, one or more additional therapeutic agents are administered prophylactically. In one aspect, compositions comprising immune cells are administered with agents for managing adverse events, many of which are described elsewhere in this application, including in 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 cardiac 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), convulsions, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0197] Examples of such agents include, but are not limited to, tocilizumab, steroids (e.g., methylprednisolone), and rabbit antithymocyte globulin. In some embodiments, vancomycin and aztreonam (1 gm each IV twice daily) may be administered for non-neutrophilic fever. In some embodiments, the method further comprises administering a non-sedating anticonvulsant for seizure prevention, administering at least one of erythropoietin, darbepoetin alfa, platelet transfusion, filgrastim, or pegfilgrastim, and / or administering tocilizumab or siltuximab. In one embodiment, the agent is a CSF family member, such as GM-CSF (granulocyte-macrophage colony-stimulating factor, also known as CSF2). GM-CSF can be produced by several hematopoietic and non-hematopoietic cell types upon stimulation and can activate / "prime" myeloid populations to produce inflammatory mediators such as TNF and interleukin-1β (IL1β). In some embodiments, the GM-CSF inhibitor is an antibody that binds to and neutralizes circulating GM-CSF. In some embodiments, the antibody is selected from lenzilumab, namilumab (AMG203), GSK3196165 / MOR103 / Otilimab (GSK / MorphoSys), KB002 and KB003 (KaloBios), MT203 (Micromet and Nycomed), and MORAb-022 / gimsilumab (Morphotek). In some embodiments, the antibody is a biosimilar thereof. 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 made by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110 / Lanotuzumab), or a biosimilar version of any one of them. In some embodiments, the inhibitor or antagonist inactivates the activity of either the GM-CSF-R-alpha (also known as CSF2R) or CSF1R receptor. In some embodiments, the inhibitor is selected from mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor alpha monoclonal antibody currently being developed by MedImmune, Inc.; cabilalizumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly), Emactuzumab, 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., heteroaryl amides, quinolinones, pyrido-pyrimidos; BLZ945 by Novartis), PLX7486, ARRY-382, pexidiltinib (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), K120227 (CAS 623142-96-1), AC708 by Ambit Biosciences, or Cannarile et al., Journal for Immuno Therapy of Cancer 2017, 5:53 and U.S. Patent Application Publication No. 20180371093 (these are incorporated by reference herein for the inhibitors they disclose. Additional neutralizing antibodies against GM-CSF or its receptor are described in the art, for example, "GM-CSF as a target in inflammatory / autoimmune disease: current evidence and future therapeutic potential," Hamilton, J.A. Expert Rev. Clin. Immunol., 2015; and "Targeting GM-CSF in inflammatory diseases," Wicks, IP, Roberts, A.W. Nat. Rev. Rheumatol., 2016. In other embodiments, the agent is an anti-IL6 or anti-IL-6 receptor blocker, including tocilizumab and siltuximab.

[0198] In one embodiment, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and ethylenimines and methylameramines, including trimethylmelamine regimes; nitrogen mustards such as chlorambucil and chlornaphazine. Chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzino Filin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5-fluorouracil (5-FU) antimetabolites such as; folic acid analogues, for example, denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, for example, calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Antiadrenergics, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamt; pi Larubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (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; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin), ONTAK™ (denileukin diftitox), esperamycin; capecitabine;and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In some embodiments, compositions comprising the CAR- and / or TCR-expressing immune effector cells disclosed herein can be administered in conjunction with antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Combinations of chemotherapeutic agents, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, are also appropriately administered. ;

[0199] The (chemo)therapeutic agent may be administered simultaneously with or within one week after administration of the engineered cells or nucleic acids. In other embodiments, the (chemo)therapeutic agent is administered 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 administration of the engineered cells or nucleic acids. In some embodiments, the (chemo)therapeutic agent is administered at least one month prior to administration of the cells or nucleic acids. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0200] A variety of additional therapeutic agents can be used in conjunction / combination with the compositions or agents / treatments described herein. For example, additional therapeutic agents that may be useful include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche), tocilizumab (with and without corticosteroids), inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R (anti-CSF1 antibodies manufactured by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110 / lacnotuzumab), mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor alpha monoclonal antibody currently in development by MedImmune, Inc.; caviralizumab (Five Prime Therapeutics; LY3022855 (IMC-CS4) (Eli Lilly), Emactuzumab, 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., heteroaryl amides, quinolinones, pyrido-pyrimidos; BLZ945 by Novartis), PLX7486, ARRY-382, pexidiltinib (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), K120227 (CAS 623142-96-1), AC708 by Ambit Biosciences, or Cannarile et al., Journal for Immuno Therapy of Cancer 2017, 5:53 and U.S. Patent Application Publication No. 20180371093, which are incorporated herein by reference for the inhibitors they disclose. Additional neutralizing antibodies against GM-CSF or its receptor are described in the art.Additional therapeutic agents suitable for use in combination with the compositions or medicaments / treatments and methods disclosed herein include ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab emtansine (KADCYL®), and ribozyme inhibitors (RIRI). A(R), imatinib (GLEEVEC(R)), cetuximab (ERBITUX(R)), panitumumab (VECTIBIX(R)), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, lenalidomide, axitinib, masitinib , pazopanib, sunitinib, sorafenib, tocilizumab, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, carbozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruki These include, but are not limited to, mTOR inhibitors such as solitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as the CDK inhibitor palbociclib.

[0201] The composition or drug / therapeutic agent containing immune cells may be administered or may be administered together with an anti-inflammatory agent. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, corticosteroids, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen, oxycodone, proporoxifene hydrochloride, and tramadol. 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 and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0202] The compositions or drugs / therapeutics described herein may be administered in conjunction with cytokines and / or cytokine regulatory agents 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); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; murelan inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-beta; platelet growth factor; transforming growth factor (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factor (IGF); Growth factor-I and growth factor-II; erythropoietin (EPO, Epogen®, Procrit®); bone morphogenetic factor; interferons, e.g., interferon-alpha, beta, and gamma; colony-stimulating factors (CSFs), e.g., macrophage-CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), e.g., IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors, e.g., TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. In one embodiment, the compositions described herein are administered in conjunction with a steroid or corticosteroid.

[0203] Corticosteroid therapy can be used to treat adverse events. Corticosteroids (or any other steroids, as well as any other treatment for adverse events) can be used prophylactically before any symptoms of the adverse event are detected and / or after the adverse event is detected. They can be administered one or more days before T cell administration, on the day of T cell administration (before, after, and / or during T cell administration), and / or after T cell administration. They can be administered before, during, or after pretreatment. Any corticosteroid can be suitable for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, the two are administered in combination. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include alclometasone, alginate, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasone, clocortolone (e.g., clocortolone pivalate), cloprednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortivazol, deflazacol, desonide, desoximetasone, dexamethasone (e.g., dexamethasone phosphate), 21, dexamethasone acetate, dexamethasone sodium phosphate), diflorasone (e.g., diflorasone diacetate), diflucortralone, difluprednate, enoxolone, furazacor, fluclonide, fludrocortisone (e.g., fludrocortisone acetate), flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, flucortine, flutrolone, fluorometholone (e.g., fluorometholone acetate), fluperolone (e.g., fluperone acetate), fluprednidene, fluprednisolone, flurandrenolide, fluticasone (e.g., fluticasone propionate), formocortal,Halcinonide, halobetasol, halometasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), paramethasone (e.g., paramethasone acetate) tazone), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulfobenzoate, prednisolone stearate, prednisolone These glucocorticoids and their salts include, but are not limited to, donisolon tebutate, prednisolone tetrahydrophthalate), prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are described in detail in, for example, Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980), and Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) or any other edition;In some embodiments, the glucocorticoid is selected from among cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In another embodiment, the steroid is a mineralocorticoid. Any other steroid may be used in the methods provided herein.

[0204] The one or more corticosteroids can be administered at any dose and frequency that can be adapted to the severity / grade of the adverse event (e.g., CRS and NE). Tables 13, 14, and 16 provide examples of dosing regimens for managing CRS and NE. In another embodiment, the corticosteroid administration comprises dexamethasone 10 mg orally or IV one to four times per day. Another embodiment, sometimes referred to as a "high-dose" corticosteroid, comprises methylprednisone 1 g / day IV, either 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.

[0205] The corticosteroid can be administered in any amount effective to ameliorate one or more symptoms associated with CRS or an adverse event such as neurotoxicity. The corticosteroid, e.g., glucocorticoid, can be administered in an amount of about 0.1 to 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 to a 70 kg adult subject, for example. Typically, corticosteroids, such as glucocorticoids, are administered to an average adult human subject in an amount of about 0.4 to 20 mg per dose, e.g., 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.

[0206] In some embodiments, the corticosteroid is administered at a dose of, for example, 0.001 or about 0.001 mg / kg (of 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, for an average adult subject, typically weighing about 70 kg to 75 kg. g, 0.08mg / kg, 0.085mg / kg, 0.09mg / kg, 0.095mg / kg, 0.1mg / kg, 0.15mg / kg, 0.2mg / kg, 0.25mg / kg, 0.30mg / kg, 0.35mg / kg, 0.40mg / kg, 0.45mg / kg, 0.50mg / kg, 0.55mg / kg, 0.60mg / kg, 0.65mg / The compound may be administered at a dose of 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.

[0207] Generally, differences in potency exist between different corticosteroids, so the dose of corticosteroid administered will depend on the individual corticosteroid. Typically, drugs vary in potency, so it is understood that doses to achieve equivalent effects may differ. Equivalents in terms of potency for various glucocorticoids and routes of administration are well known. Information regarding equivalent steroid dosages (in a non-chronotherapeutic manner) can be found in British National Formulary (BNF) 37 (March 1999).

[0208] In some embodiments, the adverse event / reaction may be selected from one or more of the following: TIFF0007813701000007.tif212148

[0209] Other adverse reactions include gastrointestinal disorders: dry mouth; infectious and parasitic disorders: fungal infections; metabolic and nutritional disorders: dehydration; nervous system disorders: ataxia, convulsions, increased intracranial pressure; respiratory, thoracic and mediastinal disorders: respiratory failure, pulmonary edema; skin and subcutaneous tissue disorders: rash; vascular disorders: bleeding.

[0210] In one embodiment, symptoms of cytokine release syndrome include, but are not limited to, fever, rigors, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxia, tachycardia, hypotension, widened 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 may be graded by the method described in Lee, et al., Blood 2014;124:188-195.

[0211] In some embodiments, adverse events are managed with tocilizumab (or another anti-IL6 / IL6R agent / antagonist) for toxicity prevention, corticosteroid therapy, or anticonvulsants. In some embodiments, adverse events are managed with one or more agents selected from inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, antithymocyte globulin, lenzilumab, mavrilimumab, cytokines, and anti-inflammatory agents.

[0212] In some embodiments, the present disclosure provides methods of preventing the occurrence or reducing the severity of adverse reactions to the T cell therapies of the present disclosure. In some embodiments, the cell therapy is administered with one or more agents that prevent, delay the onset, reduce symptoms, or treat adverse events, including cytokine release syndrome and neurotoxicity. In one embodiment, the agents are described above. In other embodiments, the agents are described below. In some embodiments, the agents are administered before, after, or simultaneously with administration of the cells, using one of the methods and dosages described elsewhere herein. In one embodiment, the agents are administered to a subject who may be predisposed to a disease but has not yet been diagnosed with the disease.

[0213] In this regard, the disclosed methods can include administering a "prophylactically effective amount" of tocilizumab, corticosteroid therapy, and / or an anticonvulsant for toxicity prevention. In some embodiments, the methods include administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, lenzilumab, mavrilimumab, a cytokine, and / or an anti-inflammatory agent. The pharmacological and / or physiological effect can be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" can refer to an amount effective to achieve a desired prophylactic result (e.g., prevention of the onset of an adverse reaction) at the dosage and for the period necessary.

[0214] In some embodiments, the method includes managing an adverse reaction in any subject. In some embodiments, the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reaction, serious infection, cytopenia, and hypogammaglobulinemia. In some embodiments, the signs and symptoms of the adverse reaction are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients have been identified and selected based on one or more biomarkers of an adverse event. In some embodiments, patients are identified and selected solely by clinical symptoms (e.g., the presence and grade of toxic symptoms). In some embodiments, adverse events are managed according to any one of the protocols in Tables 13, 14, 16, and 17.

[0215] 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 symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. Patients experiencing Grade 2 or higher CRS (e.g., hypotension unresponsive to fluids or hypoxia requiring supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, patients experiencing severe CRS should be considered for echocardiograms to assess cardiac function. In cases of severe or life-threatening CRS, intensive supportive care may be considered. In some embodiments, the method includes monitoring the patient daily for at least seven days after infusion at a certified medical facility for signs and symptoms of CRS. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for four weeks after infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention whenever signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as indicated at the first sign of CRS.

[0216] 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 experiencing grade 2 or higher neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care is provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, and anxiety.

[0217] In some embodiments, the cell therapy is administered before, during / concurrently with, and / or after administration of one or more agents (e.g., steroids) or treatments (e.g., weight loss) that treat or prevent one or more symptoms of an adverse event (prophylactic). A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and duration required. In one embodiment, a prophylactically effective amount is used in a subject prior to or at an early stage of disease. In one embodiment, a prophylactically effective amount will be less than a therapeutically effective amount. In one embodiment, the treatment or prevention of an adverse event is administered to any patient receiving, receiving, or who has received cell therapy. In some embodiments, the method of managing an adverse event includes monitoring the patient daily for at least seven days after the infusion at a certified medical facility for signs and symptoms of neurotoxicity. In some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity and / or CRS for four weeks after the infusion.

[0218] In some embodiments, the present disclosure provides two methods for managing adverse events in subjects receiving CAR T cell therapy with steroids and anti-IL6 / anti-IL-6R antibodies. In one embodiment, the present disclosure provides a method of adverse event management in which corticosteroid therapy is initiated for the management of all cases of Grade 1 CRS if all Grade ≧1 neurological events have not improved after three days. In one embodiment, the present disclosure provides a method of tocilizumab for the management of all cases of Grade 1 CRS if all Grade ≧2 neurological events have not improved after three days. In one embodiment, the present disclosure provides a method of reducing total steroid exposure in patients undergoing adverse event management after CAR T cell administration, the method comprising initiating corticosteroid therapy for the management of all cases of Grade 1 CRS if all Grade ≧1 neurological events have not improved after three days, and / or initiating tocilizumab for all cases of Grade 1 CRS if all Grade ≧2 neurological events have not improved after three days. In one embodiment, the corticosteroid and tocilizumab are administered in a regimen selected from those exemplified in the Examples section. In one embodiment, the present disclosure provides that early steroid use is not associated with an increased risk of severe infection, decreased CAR T cell expansion, or decreased tumor response.

[0219] In one embodiment, the present disclosure supports the safety of levetiracetam prophylaxis 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. Accordingly, in one embodiment, the present disclosure provides a method for managing adverse events in patients treated with CAR T cells, comprising administering a prophylactic dose of an anticonvulsant to the patient. In some embodiments, the patient is administered levetiracetam (e.g., 750 mg orally or intravenously twice daily) starting on day 0 of CAR T cell therapy (post-conditioning) and, if a neurological event occurs after discontinuation of prophylactic levetiracetam, at the onset of Grade ≥ 2 neurotoxicity. In one embodiment, if the patient does not experience any Grade ≥ 2 neurotoxicity, levetiracetam is tapered and discontinued as clinically indicated. In one embodiment, levetiracetam prophylaxis is combined with any other adverse event management protocol.

[0220] In one embodiment, patients may receive levetiracetam (750 mg orally or intravenously, twice daily) starting on day 0. At the onset of a grade ≥ 2 neurological event, the levetiracetam dose is increased to 1000 mg twice daily. If the patient does not experience any grade ≥ 2 neurological events, levetiracetam is reduced and discontinued as clinically indicated. Patients also receive tocilizumab (8 mg / kg IV over 1 hour [not to exceed 800 mg]) on day 2. Additional tocilizumab (± corticosteroids) may be recommended at the onset of grade 2 CRS in patients with comorbidities or elderly patients, or otherwise in the case of grade ≥ 3 CRS. Tocilizumab is initiated for patients experiencing a grade ≥2 neurological event, and corticosteroids are added for patients with comorbidities or advanced age, or in the presence of any grade ≥3 neurological event that worsens despite tocilizumab use.

[0221] In one embodiment, the present disclosure provides that the use of prophylactic steroids appears to reduce the rate of severe CRS and NE to a similar extent as initial steroid administration. Accordingly, the present disclosure provides a method for adverse event management in CAR T-cell therapy, in which patients receive dexamethasone 10 mg PO on days 0 (pre-infusion), 1, and 2. Steroids may be administered starting with grade 1 NE and grade 1 CRS when there is no improvement after 3 days of supportive care. Tocilizumab may be administered for grade ≥ 1 CRS when there is no improvement after 24 hours of supportive care. In one embodiment, the present disclosure provides adverse event management of CAR T-cell therapy with an antibody that neutralizes and / or depletes GM-CSF, preventing or reducing treatment-related CRS and / or NE in treated patients. In one embodiment, the antibody is lenzilumab.

[0222] In some embodiments, the adverse events are managed by administering an agent that is an antagonist or inhibitor of IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the agent is an antibody that neutralizes IL-6 activity, e.g., an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the agent is or includes an anti-IL-6R antibody such as tocilizumab (atlizumab) or sarilumab. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Patent No. 8,562,991. In some embodiments, the agent targeting IL-6 is an anti-IL-6 antibody, e.g., siltuximab, elcilimomab, ALD518 / BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), and combinations thereof. In some embodiments, the agent may neutralize IL-6 activity by inhibiting ligand-receptor interaction. In some embodiments, the IL-6 / IL-6R antagonist or inhibitor is an IL-6 mutein, such as those described in U.S. Patent No. 5,591,827. In some embodiments, the agent that is an IL-6 / IL-6R antagonist or inhibitor is a small molecule, a protein or peptide, or a nucleic acid.

[0223] In some embodiments, other agents that can be used to manage adverse reactions and their symptoms include cytokine receptor or cytokine antagonists or inhibitors. In some embodiments, the cytokine or receptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R / CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFalpha, TNFR1, e.g., TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-beta receptor (TGF-βI, II, or III), IFN-gamma receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFalpha receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), IL-1 and IL-1Ralpha / IL-1beta. In some embodiments, the agent comprises cituximab, sarilumab, olokizumab (CDP6038), elcilimomab, ALD518 / BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the agent is a cytokine antagonist or inhibitor, for example, transforming growth factor beta (TGF-beta), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNF-alpha, IL-1, interferon gamma (IFN-gamma), or monocyte chemoattractant protein-I (MCP-1). In some embodiments, the compound targets (e.g., inhibits or is an antagonist of) a cytokine receptor, such as IL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-β receptor (TGF-βI, II, or III), IFN-γ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), and combinations thereof.In some embodiments, the agent is administered before, after, or simultaneously with administration of the cells, by one of the methods and doses described elsewhere herein.

[0224] In some embodiments, the agent is administered at a dosage of 1 mg / kg to 10 mg / kg (or about 1 mg / kg to 10 mg / kg), 2 mg / kg to 8 mg / kg (or about 2 mg / kg to 8 mg / kg), 2 mg / kg to 6 mg / kg (or about 2 mg / kg to 6 mg / kg), 2 mg / kg to 4 mg / kg (or about 2 mg / kg to 4 mg / kg), or 6 mg / kg to 8 mg / kg (or about 6 mg / kg to 8 mg / kg) (each inclusive), or the agent is administered at a dosage of at least about, or about, 2 mg / kg, 4 mg / kg, 6 mg / kg, or 8 mg / kg. In some embodiments, the agent is administered at a dosage of about 1 mg / kg to 12 mg / kg, e.g., 10 mg / kg, 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, such as tocilizumab in particular, is administered before, after, or simultaneously with administration of the cells, by one of the methods and dosages described elsewhere herein.

[0225] In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, it can be managed according to the recommendations of Protocol A, which can also be used in combination with other therapies disclosed herein, such as neutralization or reduction of the CSF / CSFR1 axis. Patients experiencing Grade 2 or higher CRS (e.g., hypotension unresponsive to fluids or hypoxia requiring supplemental oxygen) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, patients experiencing severe CRS should be considered for echocardiograms to assess cardiac function. In severe or life-threatening CRS, intensive supportive care may be considered. In some embodiments, a tocilizumab biosimilar or its equivalent may be used instead of tocilizumab in the methods disclosed herein. In other embodiments, another anti-IL6R agent may be used instead of tocilizumab.

[0226] In some embodiments, adverse events are managed according to the following protocol (Protocol A). TIFF0007813701000008.tif124162(a)Lee DW et al. (2014).Current concepts in the diagnosis and management of cytokine release syndrome.Blood.2014 Jul 10;124(2):188-195. (b) See Table 2 for management of neurotoxicity. (c) For more information, see ACEMTRA® (tocilizumab) prescribing information, https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). First US approval noted in 2010. Neurotoxicity

[0227] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients experiencing grade 2 or higher neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, provide intensive supportive care. Consider a non-sedating anti-seizure medication (e.g., levetiracetam) for seizure prevention in any grade 2 or higher neurotoxicity. The following treatments can be used in combination with other treatments of the present disclosure, such as neutralization or reduction of the CSF / CSFR1 axis.

[0228] In some embodiments, adverse events are managed according to the following protocol (Protocol B). TIFF0007813701000009.tif108163

[0229] Additional safety management strategies with corticosteroids

[0230] Administration of corticosteroids and / or tocilizumab in Grade 1 can be considered prophylactic. Supportive care can be provided in all protocols for all CRS and NE severity grades. In one embodiment of the protocol for managing adverse events associated with CRS, tocilizumab, and / or corticosteroids, the following is administered: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only if comorbidities or elderly); and / or corticosteroids (only if comorbidities or elderly); Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids. In another embodiment of the protocol for managing adverse events associated with CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: tocilizumab (if no improvement after 3 days); and / or corticosteroids (if no improvement after 3 days); Grade 2 CRS: tocilizumab; and / or corticosteroids; Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids, high dose.

[0231] In one embodiment of a protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following are administered: Grade 1 NE: no tocilizumab; no corticosteroids; Grade 2 NE: no tocilizumab; no corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids (standard dose only if no improvement to tocilizumab); Grade 4 NE: tocilizumab; and / or corticosteroids. In another embodiment of a protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following are administered: Grade 1 NE: no tocilizumab; and / or corticosteroids; Grade 2 NE: tocilizumab; and / or corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids, high dose; Grade 4 NE: tocilizumab; and / or corticosteroids, high dose. In one embodiment, corticosteroid treatment is initiated for CRS of grade 2 or higher, and tocilizumab treatment is initiated for CRS of grade 2 or higher. In one embodiment, corticosteroid treatment is initiated for CRS of grade 1 or higher, and tocilizumab treatment is initiated for CRS of grade 1 or higher. In one embodiment, corticosteroid treatment is initiated for NE of grade 3 or higher, and tocilizumab treatment is initiated for CRS of grade 3 or higher. In one embodiment, corticosteroid treatment is initiated for CRS of grade 1 or higher, and tocilizumab treatment is initiated for CRS of grade 2 or higher. In some embodiments, prophylactic use of tocilizumab administered on day 2 may reduce the rate of CRS of grade 3 or higher. One or more corticosteroids may be administered at any dose and frequency that can be adapted to the severity / grade of the adverse event (e.g., CRS and NE). Tables 1 and 2 provide examples of dosing regimens for managing CRS and NE, respectively. In another embodiment, the corticosteroid administration comprises dexamethasone 10 mg orally or IV one to four times per day. Another embodiment, sometimes referred to as "high-dose" corticosteroids, comprises methylprednisone 1 g / day IV, either alone or in combination with dexamethasone.In some embodiments, one or more corticosteroids are administered at a dose of 1-2 mg / kg per day. Generally, the corticosteroid dose administered depends on the specific corticosteroid, as differences in potency exist between different corticosteroids. It is understood that drugs typically vary in potency, resulting in different doses for equivalent effects. Potency-equivalent amounts for various glucocorticoids and routes of administration are well known. Information regarding equivalent steroid doses (non-chronotherapeutic) can be found in British National Formulary (BNF) 37, March 1999. The present application provides dosage and administration of cells prepared by the methods of the present application. For example, an infusion bag for CD19-directed genetically modified autologous T-cell immunotherapy contains approximately 68 mL of a suspension of chimeric antigen receptor (CAR)-positive T cells for infusion. In some embodiments, the CAR T cells are formulated into approximately 40 mL for infusion. In some embodiments, the CAR T cell product is formulated in a total volume of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900, 1000 mL. In one aspect, the dose and administration of cells prepared by the methods of the present application, for example, an infusion bag of CD19-directed genetically modified autologous T cell immunotherapy, is 1 x 10 in about 40 mL. 6 The target dose is approximately 1 x 10 per kg of body weight. 6 ~about 2×10 6 CAR-positive viable T cells, up to 2 x 10 8 It may be a CAR-positive viable T cell.

[0232] In some embodiments, the dosage form comprises a cell suspension for infusion in single-use patient-specific infusion bags, the route of administration is intravenous, and the entire contents of each single-use patient-specific bag is infused over 30 minutes by gravity or a peristaltic pump. In one embodiment, the dosing regimen is 2.0 x 10 6 anti-CD19 CAR T cells / kg body weight (±20%), maximum dose 2 × 10 8anti-CD19 CAR T cells (for subjects >= 100 kg). In some embodiments, the T cells comprising the dose are CD19 CAR-T cells.

[0233] 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™ (YESCARTA®, axicabtagene ciloleucel), which is prepared by one of the methods of this application. The amount of CAR T cells, dosing regimen, method of administration, subject, and cancer within these methods are described elsewhere in this application, alone or in combination with another chemotherapeutic agent, with or without prior treatment, for any of the patients described elsewhere in this application.

[0234] The following examples are intended to illustrate various aspects of the present application. Accordingly, the specific aspects discussed should not be construed as limitations on the scope of the present application. For example, although the following examples are directed to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), those skilled in the art will understand that the methods described herein can be applied to immune cells transduced with any CAR. Those skilled in the art will understand that various equivalents, changes, and modifications can be made without departing from the scope of the present application, and that such equivalent aspects are encompassed herein. Furthermore, all references cited in this application are incorporated herein by reference in their entirety, as if fully set forth herein.

[0235] The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, dictionaries, documents, manuscripts, genome database sequences, and scientific literature cited herein are incorporated herein by reference.

[0236] Other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings and examples. [Example]

[0237] Example 1

[0238] In this trial, patients with R / R MCL who had received one to five prior therapies, including a Bruton's tyrosine kinase inhibitor (BTKi), were treated with autologous anti-CD19 CAR-T cells.

[0239] Eligible patients (≥18 years of age) with R / R MCL had an ECOG score of 0-1 and had received ≤5 prior therapies, including chemotherapy, anti-CD20 antibodies, and BTK inhibitors (BTKi). Patients were randomly assigned to receive leukapheresis and chemotherapy (cyclophosphamide 300 mg / m 2 / d and fludarabine 30 mg / m 2 / d, 3 days) and then 2 × 10 6Patients received infusions of CD19 CAR-T at a target dose of 10 CAR T cells / kg. Patients could have received bridging therapy with dexamethasone, ibrutinib, or acalabrutinib after leukapheresis and before chemotherapy. The primary endpoint was the objective response rate (ORR [complete response (CR) + partial response (PR)]) according to the Lugano classification. The interim efficacy endpoint was investigator-assessed using the modified IWG response criteria for malignant lymphoma. Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), OS, frequency of adverse events (AEs), blood CAR T cell levels, and serum cytokine levels.

[0240] Twenty-eight patients received CD19 CAR-T cells and were followed for more than 1 year (median 13.2 months [range, 11.5-18.5]). 43 percent of patients had an ECOG score of 1, 21% had blastoid morphology, 82% had stage IV disease, 50% had intermediate / high-risk MIPI, 86% had received a median of four prior therapies (range, 1-5), and 57% were refractory to their most recent prior therapy. In 20 of 28 patients, the median Ki-67 index was 38% (range, 5%-80%). Eight patients received bridging therapy, and all had disease after bridging. The ORR was 86% (95% CI, 67%-96%), and the CR rate was 57% (95% CI, 37%-76%). Seventy-five percent of responders had ongoing responses, and 64% of treated patients had ongoing responses. The 12-month estimates for DOR, PFS, and OS were 83% (95% CI, 60% to 93%), 71% (95% CI, 50% to 84%), and 86% (95% CI, 66% to 94%), respectively, with median OS not reached. Grade ≥3 AEs (occurring in ≥20% of patients) were anemia (54%), thrombocytopenia (39%), neutropenia (36%), neutrophil count decreased (32%), leukocyte count decreased (29%), encephalopathy (25%), and hypertension (21%). Grade 3 / 4 cytokine release syndrome (CRS), as assessed by Lee DW, et al. Blood 2014;124:188, was reported in 18% of patients and manifested as hypotension (14%), hypoxia (14%), and fever (11%). Grade 3 / 4 neurological events (NE) were reported in 46% of patients and included encephalopathy (25%), confusion (14%), and aphasia (11%). No grade 5 CRS or NE occurred. All CRS events and most NE (15 / 17 patients) were reversible. The median time to onset and resolution of CRS was 2 days (range, 1-7) and 13 days (range, 4-60), respectively. The median time to onset of NE was 6 days (range, 1-15), and the median time to resolution was 20 days (range, 9-99).The median CAR T cell levels, measured by peak and area under the curve, were 99 cells / μL (range, 0.4-2589) and 1542 cells / μL (range, 5.5-27239), respectively. Peak CAR T cell proliferation was observed between days 8 and 15 and decreased over time. Example 2

[0241] This example provides additional analysis for the above study. Eligible patients were aged 18 years or older, had pathologically confirmed MCL with evidence of either cyclin D1 overexpression or the presence of t(11;14), and had relapsed / refractory disease to one to five prior regimens for MCL. Prior therapy must have included anthracycline- or bendamustine-containing chemotherapy, an anti-CD20 monoclonal antibody, and ibrutinib or acalabrutinib. All patients received prior BTK therapy. Patients must have received prior BTK therapy, but it did not have to be their most recent therapy prior to study entry, and patients did not need to be resistant to BTK therapy. Eligible patients had an absolute lymphocyte count ≥ 100 / μL. Patients who underwent autologous SCT within 6 weeks of CD19 CAR-T infusion or previous CD19-targeted therapy or allogeneic SCT were excluded.

[0242] Additional inclusion criteria included the following: at least one measurable lesion. Previously irradiated lesions were considered measurable only if there was evidence of progression after completion of radiotherapy; if the only measurable disease was lymph node disease, at least one lymph node should be ≥2 cm; magnetic resonance imaging (MRI) of the brain showing no evidence of central nervous system (CNS) lymphoma; at least 2 weeks since any prior systemic therapy or BTKi (ibrutinib or acalabrutinib) at the time the patient was scheduled for leukapheresis, with the exception of systemic inhibitory / stimulatory immune checkpoint therapy. At the time the patient is scheduled for leukapheresis, at least three half-lives must have elapsed since any prior systemic inhibitory / stimulatory immune checkpoint molecular therapy (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists); toxicities from prior therapy must be stable and resolved to Grade 1 or less (excluding clinically unrelated toxicities, e.g., alopecia); Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; absolute neutrophil count (ANC) ≥ 1000 / μL; platelet count ≥ 75000 / μL; absolute lymphocyte count ≥ 100 / μL; adequate renal, hepatic, pulmonary, and cardiac function as defined as follows: Creatinine clearance (estimated by the Cockcroft-Gault formula) ≥ 60 cc / min; serum alanine aminotransferase / aspartate aminotransferase ≤ 2.5 upper limit of normal (ULN); total bilirubin ≤ 1.5 mg / dL (excluding patients with Gilbert syndrome); cardiac ejection fraction ≥ 50%, no evidence of pericardial effusion as determined by echocardiography (ECHO), no clinically relevant electrocardiogram (ECG) findings; no clinically relevant pleural effusion; baseline oxygen saturation > 92% on room air; and women of childbearing potential must have a negative urine or serum pregnancy test. Women who were surgically sterilized or postmenopausal for at least 2 years were considered non-childbearing potential.

[0243] Additional exclusion criteria included the following: a history of malignancy other than nonmelanoma skin cancer or carcinoma in situ (e.g., cervical, bladder, breast) unless there was a disease-free interval of at least 3 years; a history of allogeneic stem cell transplant; prior CAR therapy or other genetically modified T-cell therapy; a history of severe immediate hypersensitivity reactions due to aminoglycosides; the presence of fungal, bacterial, viral, or other infections that were uncontrolled or required intravenous (IV) antibiotics for management. Uncomplicated urinary tract infections (UTIs) and uncomplicated bacterial pharyngitis were allowed after consultation with a medical monitor if they responded to active treatment; and a history of human immunodeficiency virus (HIV) infection or acute or chronic active hepatitis B or C infection. Patients with a history of hepatitis infection must have had the infection cleared as determined by standard serologic and genetic testing; presence of an indwelling line or drain (e.g., percutaneous nephrostomy tube, indwelling Foley catheter, biliary drain, or pleural / peritoneal / pericardial catheter). Ommaya reservoirs and dedicated central venous access catheters, e.g., Port-a-Cath or Hickman catheters, were permitted.Patients with detectable cerebrospinal fluid malignant cells or brain metastases, or a history of CNS lymphoma, cerebrospinal fluid malignant cells, or brain metastases; a history or presence of CNS disorders, such as seizure disorders, cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, reversible leukoencephalopathy, or any autoimmune disease involving the CNS; a history of myocardial infarction, cardiac angioplasty or stent placement, unstable angina, active arrhythmia, or other clinically relevant cardiac disease within 12 months of enrollment; patients with atrial or ventricular lymphoma involvement; symptomatic deep vein thrombosis within the last 6 months of enrollment history of severe immediate allergic reaction to any of the drugs used in this study; receipt of a live vaccine within 6 weeks prior to the start of the planned conditioning regimen; women of childbearing potential who are pregnant or breastfeeding due to the potential adverse effects of preparatory chemotherapy on the fetus or infant; male and female patients who are not willing to practice contraception from the time of consent until 6 months after completion of CD19 CAR-T cell therapy; patients who, in the investigator's judgment, are unlikely to complete all protocol-required study visits or procedures, including follow-up visits, or to comply with study requirements for participation; and a history of an autoimmune disease (e.g., Crohn's disease, rheumatoid arthritis, systemic erythematosus) within the last 2 years that has resulted in end-organ damage or requires systemic immunosuppression / systemic disease-modifying agents.

[0244] All patients underwent leukapheresis to obtain cells for the manufacturing of CD19 CAR-T cell therapy. + / CD8 + Modified axicabtagene ciloleucel for the elimination of circulating lymphoma cells by positive enrichment of cells. Fludarabine (30 mg / m 2 / day) and cyclophosphamide (500 mg / m 2 / day) on days -5, -4, and -3, followed by 2 x 10 6A single intravenous infusion of CD19 CAR-T cells was administered at CAR T cells / kg. Dosing was informed by studies of axicabtagene ciloleucel in large B-cell lymphoma and CD19 CAR-T cells in acute lymphoblastic leukemia. Neelapu SS et al. The New England journal of medicine 2017;377:2531, Locke FL et al. Mol Ther 2017;25:285, Shah BD et al. Journal of Clinical Oncology 2019;37:(suppl;abstr 7006), and Lee DW et al. Annals of oncology: official journal of the European Society for Medical Oncology / ESMO 2017;28:1008PD, all of which are incorporated herein by reference in their entireties. After leukapheresis and before conditioning, patients with a high disease burden could receive bridging therapy with dexamethasone or equivalent corticosteroids, ibrutinib, or acalabrutinib at the investigator's discretion, followed by a repeat baseline positron emission tomography-computed tomography (PET-CT) scan. The goal of bridging therapy was not curative but to keep patients stable for the duration of the treatment period. Hospitalization after CD19 CAR-T cell infusion was required up to day 7.

[0245] The primary endpoint was the objective response rate (ORR [complete response (CR) + partial response (PR)]) assessed by an independent radiological review committee (IRRC) using the Lugano classification (Chesson et al., J Clin Oncol 2014;32:3059-68). Bone marrow evaluation was required in addition to PET-CT to confirm CR. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), OS, investigator-assessed ORR according to Cheson et al., J Clin Oncol 2007;25:579-86, incidence of adverse events (AEs), blood CAR T cell and serum cytokine levels, and change in score over time on the European Quality of Life-5 Dimensions with 5 levels per dimension (EQ-5D-5L). The presence, proliferation, and persistence of CAR T cells and serum cytokines, as well as their association with clinical outcomes, were assessed as previously reported. Kochenderfer JN et al. J Clin Oncol 2017;35:1803-13, Locke FL et al. Mol Ther 2017;25:285-95, both of which are incorporated by reference in their entireties.

[0246] Changes in EQ-5D-5L scores from baseline to 6 months were assessed. Cytokine release syndrome (CRS) was graded according to Lee et al. Blood 2014;124:188, which is incorporated herein by reference in its entirety. AE severity, including neurological events and CRS symptoms, was graded using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Minimal residual disease (MRD, sensitivity 10 -5) was an exploratory analysis evaluated in cryopreserved peripheral blood mononuclear cells at baseline and 1, 3, and 6 months and analyzed by next-generation sequencing using the clonoSEQ assay (Adaptive Biotechnologies, Seattle, WA).

[0247] For all patients, positron emission tomography-computed tomography (PET-CT) scans of disease-specific regions were required at baseline, 4 weeks postinfusion, and periodically during the posttreatment period. Bone marrow aspirates / biopsies were required to confirm complete responses in patients with bone marrow involvement at baseline and in patients with indeterminate bone marrow involvement at baseline, or results were unavailable if a baseline bone marrow biopsy was not performed. Patients with symptoms of CNS malignancy underwent lumbar puncture at screening for cerebrospinal fluid (CSF) analysis. Lumbar punctures were also performed, if applicable, in patients with new-onset grade ≥2 neurotoxicity after anti-CD19 CAR T-cell infusion. Additionally, for patients who signed the consent form, lumbar punctures for CSF collection were performed at baseline before anti-CD19 CAR T-cell infusion and at days 5 ± 3 after anti-CD19 CAR T-cell infusion, and samples were submitted to a central laboratory for analysis of changes in cytokine levels.

[0248] Sixty patients were enrolled and treated, and the primary efficacy analysis was performed after 6 months of evaluation following the Week 4 disease assessment as required by the protocol. This analysis had 96% or greater power to distinguish between active treatment with a 50% true response rate and treatment with a 25% or lower response rate, using a one-sided alpha level of 0.025. ORR was analyzed using an exact binomial test. All efficacy endpoints, including time-to-event endpoints, were analyzed in the 60 efficacy-evaluable patients using Kaplan-Meier estimates. Safety analyses were performed in all treated patients (n=68). Associations between outcomes and CAR T cell and cytokine levels were measured using the Wilcoxon rank-sum test, and P values ​​were adjusted using the Holm method. Full analysis set (N=74): This consisted of all enrolled / leukapheresis-treated patients and was used for patient dissection summaries. Safety analysis set (n=68): Defined as all patients treated with any dose of anti-CD19 CAR T cells. This analysis set was used for summary demographic and baseline characteristics and all safety analyses. Inferential analysis (efficacy-evaluable) set (n=60): Consisting of the first 60 CD19 CAR-T cell-treated patients. This analysis set was used for hypothesis testing of the primary endpoint of objective response rate at the time of the primary analysis and all other efficacy analyses. The primary endpoint hypothesis was that the ORR for CD19 CAR-T cells using central review would be greater than the pre-specified historical control rate of 25% using an exact binomial test at a one-sided significance level of 0.025. This hypothesis was tested in the inferential analysis set. The historical control rate for ORR was predetermined based on two retrospective studies that were publicly available at the time of the study protocol. These two trials evaluated outcomes after salvage therapy in patients with relapsed / refractory MCL who had progressed after BTKi treatment (prior therapy required for study eligibility). These trials showed that patients with relapsed / refractory MCL who had received three or more prior therapies before receiving BTKi had an ORR to salvage therapy of approximately 25%.Wang M et al. Lancet 2018;391:659, Martin P et al. Blood 2016;127:1559, both of which are incorporated by reference in their entireties.

[0249] Seventy-four patients were enrolled, 71 of whom received CD19 CAR-T cells, and 68 received them. The primary efficacy analysis, performed after 60 patients had been treated, demonstrated a 93% ORR (67% complete response). At a median follow-up of 12.3 months (range, 7.0-32.3 months), 57% of patients remained in remission, and the median duration of response was not reached. Estimated 12-month progression-free survival and overall survival rates were 61% and 83%, respectively. Common grade ≥3 adverse events were cytopenias (94%) and infections (32%). Grade ≥3 cytokine release syndrome and neurologic events occurred in 15% and 31%, respectively, with no fatalities. Two grade 5 infectious adverse events occurred.

[0250] CD19 CAR-T cells were manufactured for 71 patients (96%) and administered to 68 patients (92%). The median time from leukapheresis to delivery of CD19 CAR-T cells to the study site was 16 days (range, 11–128). One patient who received CD19 CAR-T cells was treated with bendamustine-rituximab due to rapid PD after leukapheresis, which made him ineligible for the study. After later progression to PD, the patient's original product was shipped from the manufacturing facility 127 days after the date of initial leukapheresis and arrived at the treatment site 1 day later. Three patients with manufacturing issues did not proceed to additional apheresis due to AEs (n=1, deep vein thrombosis), death from progressive disease (PD, n=1), or consent withdrawal (n=1). Two additional patients discontinued treatment before conditioning chemotherapy due to death from PD. One patient with persistent atrial fibrillation after receiving conditioning chemotherapy, a study exclusion criterion, was deemed ineligible for CD19 CAR-T cell infusion. The median follow-up for efficacy-evaluable patients was 12.3 months (range, 7.0-32.3), with 28 patients having a follow-up period of 24 months or longer.

[0251] The median age was 65 years (range, 38-79), and 57 patients (84%) were men (Table 1). Sixty-five percent had an ECOG performance status score of 0, and 35% had a score of 1. Patients had high-risk features at baseline, including stage IV disease (85%), blastoid or pleomorphic morphology (31%), Ki-67 proliferation index ≥ 30% (40 / 49 [82%]) (Wang ML et al. The Lancet Oncology 2016;17:48), and TP53 mutations (6 / 36 [17%]). Eighty-one percent of patients had received three or more prior lines of therapy (median, 3 [range, 1-5]).

[0252] TIFF0007813701000010.tif255158 * Exclude bone marrow and splenic involvement. † At diagnosis. ††One patient was reported by the investigator to have kappa light chain-restricted MCL at the time of diagnosis. Morphology was reported as unknown for 10 patients. § Ki-67 data were available for 49 patients at the time of diagnosis. ¶ Induction + consolidation / maintenance and / or all treatments occurring during ongoing complete response were counted as one regimen. BTKi, Bruton's tyrosine kinase inhibitor; ECOG, Eastern Cooperative Oncology Group; MCL, mantle cell lymphoma; MIPI, Mantle Cell Lymphoma International Prognostic Index; SCT, stem cell transplant.

[0253] All patients had progressed on BTKi (ibrutinib n = 58, acalabrutinib n = 16, both n = 6), and 43% had received upfront autologous SCT (Table 2). The median time from the end of the last BTKi therapy, excluding bridging, to CD19 CAR-T cell infusion was 88 days (range, 25-1047). Forty percent of patients were refractory to their most recent therapy, including three ibrutinib-intolerant patients who progressed after their most recent therapy. Twenty-five patients (37%) received bridging therapy with ibrutinib (n = 14), acalabrutinib (n = 5), dexamethasone (n = 12), and / or methylprednisolone (n = 2). Post-bridging scans showed that most patients had tumor burdens higher than the median at screening.

[0254] TIFF0007813701000011.tif51158BTKi, Bruton's tyrosine kinase inhibitor;

[0255] At a minimum follow-up of 7 months, the IRRC-assessed ORR in 60 protocol-specific patients treated with CD19 CAR-T was 93% (95% CI, 84-98), with a CR rate of 67% and a PR rate of 27%. High concordance (95%) was observed between the IRRC-assessed and investigator-assessed ORRs (Table 3).

[0256] Table 3. Response in efficacy-evaluable patients based on investigator assessment according to Cheson BD et al. J Clin Oncol 2007;25:57 and in intent-to-treat patients based on IRRC review according to the Lugano classification (2014). TIFF0007813701000012.tif63163 * No evaluation during analysis. † Concordance rate is the percentage of subjects in whom the IRRC-assessed readings concordant with the investigator-assessed readings. CR, complete response; IRRC, independent radiological review committee; N / A, not applicable; ORR, objective response rate.

[0257] For all enrolled patients (n=74), the ORR as assessed by IRRC was 85% (95% CI, 75-92), with a CR rate of 59%. ORR was consistent across key subgroups, including age, relapsed / refractory subgroup, number of prior therapies, MCL morphology, disease stage, extranodal disease, bone marrow involvement, abbreviated MIPI, CD19 positivity, tumor burden, serum lactate dehydrogenase level, TP53 mutation status, Ki-67 index, use of tocilizumab or steroids for AE management, and use of bridging therapy. The median time to initial response was 1.0 month (range, 0.8-3.1), and the median time to CR was 3.0 months (range, 0.9-9.3). Of the 42 patients who initially achieved PR or SD, 24 patients (57%) subsequently converted to CR after a median of 2.2 months (range, 1.8-8.3 months) after initial response, including 21 initial PRs and 3 initial SDs. 18 of these 24 patients remained in remission. MRD analysis was performed in 29 / 60 patients (48%). 24 / 29 patients (83% [19 CRs, 5 PRs]) were MRD-negative at week 4, and 15 / 19 patients (79%) with available data remained MRD-negative at 6 months. MRD could not be assessed in all patients because formalin-fixed, paraffin-embedded tumor biopsy samples were not available for calibration. Calibration, required by this technique, was used to establish the predominant rearranged IgH (VDJ or DJ), IgK, or IgL receptor gene sequence, which was tracked over time in the blood. Two patients who progressed after responding to CD19 CAR-T cells received a second infusion approximately 1 year and 2.6 years after the first infusion, and analysis of these patients is ongoing.

[0258] The median DOR was not reached (8.6, NE) after a median follow-up of 12.3 months (median (95% CI)). The median progression-free survival (95% CI) was not reached (9.2, NE). The median overall survival (95% CI) was also not reached (24.0, NE). Fifty-seven percent of all patients and 78% of patients in CR remained in remission. However, of the first 28 patients treated, with a median follow-up of 27.0 months (range, 25.3-32.3), 43% remained in remission without further treatment. Ongoing response rates were consistent across key covariates, including age, MCL morphology, relapsed / refractory subgroup, Ki-67 index, disease stage, extranodal disease, bone marrow involvement, abbreviated MIPI, TP53 mutation, CD19 positivity, bridging therapy, tumor burden, and use of tocilizumab or steroids. Three patients with CD19 tumors at baseline achieved a CR and continued to respond at the time of data cutoff. Median PFS and OS were not reached, with 12-month estimated rates of 61% (95% CI, 45-74) and 83% (95% CI, 71-91), respectively. Despite limited sample size, subgroup analysis of PFS showed that 6-month PFS rates were consistent among patients with blastoid or pleomorphic morphology, TP53 mutation, or Ki-67 index ≥ 50%. At the time of this analysis, 76% of all patients were alive. Of the patients who had a response, 14 had PD. One patient who had a PR underwent allogeneic SCT.

[0259] This study demonstrated a 93% ORR in 60 protocol-identified patients with relapsed / refractory MCL, all of whom relapsed after or were resistant to BTKi therapy. This ORR included a 67% CR rate after a single infusion. After a mean follow-up of 12.3 months, the median DOR was not reached, with 57% of all patients and 78% of CR patients remaining in response. The 28 patients treated with CD19 CAR-T cells had a longer median follow-up of 27 months (range 25.3-32.3 months), with 43% remaining in remission without further therapy. Response rates, including ongoing responses, were generally similar across key subgroups, including patients with high-risk features. Patients with Ki-67 ≥ 50% and blastoid / pleomorphic morphology or TP53 mutations had high ORR and 6-month PFS rates similar to the overall population, suggesting that CD19 CAR-T cell therapy may benefit patients who typically have a poor prognosis.

[0260] All patients who responded after CAR T-cell infusion achieved T-cell proliferation. This proliferation was not observed in non-responders, suggesting that this response may be related to sufficient CAR T-cell proliferation. As in previous studies, CAR T-cell levels correlated with ORR during the first 28 days, suggesting that higher proliferation resulted in a better and possibly deeper response, as indicated by >80-fold higher peak / AUC CAR T-cell levels in MRD-negative patients compared with MRD-positive patients. Response rates were also similar regardless of whether bridging therapy was administered, and most patients (87%) who underwent post-bridging scans had an increase in SPD compared with pre-bridging scans.

[0261] All treated patients experienced at least one AE of any grade, with 99% experiencing grade 3 or higher AEs (Table 2). The most common AEs of any grade were fever (94%), neutropenia (87%), thrombocytopenia (74%), and anemia (68%). The most common grade ≥3 AEs were neutropenia (85%), thrombocytopenia (51%), anemia (50%), and infection (32%). Twenty-six percent of patients had grade ≥3 cytopenia present >90 days after CD19 CAR-T cell therapy, including neutropenia (16%), thrombocytopenia (16%), and anemia (12%). CRS occurred in 91% of patients (Table 4). No patient deaths were due to CRS. Most cases were grade 1 / 2 (76%), and grade ≥3 CRS occurred in 15% of patients. The most common grade ≥3 CRS symptoms were hypotension (22%), hypoxia (18%), and fever (11%). For CRS management, 59% of patients received tocilizumab, 22% required steroids, and 16% required vasopressors. After infusion, the median time to onset of any grade and grade ≥3 CRS was 2 days (range, 1-13) and 4 days (range, 1-9), respectively, and all events resolved within a median of 11 days.

[0262] TIFF0007813701000013.tif218162 * This includes adverse events occurring in ≥30% of patients, and symptoms of CRS and neurological events occurring in ≥15% of patients. † The percentage of CRS lines was calculated for the 62 patients who experienced CRS.

[0263] Sixty-three percent of patients experienced NE (Table 4). No patient deaths were attributed to NE. Grade 1 / 2 NE occurred in 32% of patients, and grade ≥3 NE in 31%. Common grade ≥3 NE events were encephalopathy (19%), confusion (12%), and aphasia (4%). One patient developed grade 4 cerebral edema and fully recovered with aggressive multimodality treatment, including ventriculostomy. Tocilizumab and steroids were used to treat NE in 26% and 38% of patients, respectively. The median time to onset of any grade and grade ≥3 NE was 7 days (range, 1-32) and 8 days (range, 5-24), respectively. The median duration of NE was 12 days, and events resolved completely in 37 of 43 patients (86%). At the time of this analysis, events in four patients were ongoing, including grade 1 tremor (n=3), grade 2 impaired concentration (n=1), and grade 1 paresthesia (n=1). Serious AEs occurred in 68% of patients (Table 5).

[0264] TIFF0007813701000014.tif85162

[0265] Thirty-two percent of patients experienced a grade ≥ 3 infection, the most common being pneumonia (9%) (Table 6).

[0266] TIFF0007813701000015.tif90163 * One patient died from staphylococcal bacteremia, and one patient died from organizing pneumonia (who developed acute kidney injury during the infection and was found at autopsy to have previously undiagnosed pulmonary embolism in addition to organizing pneumonia).

[0267] Grade 2 cytomegalovirus infection occurred in two patients (3%). Grade 3 hypogammaglobulinemia and grade 3 tumor lysis syndrome occurred in one patient each (1%). Twenty-two patients (32%) received intravenous immunoglobulin therapy. No cases of replication-competent retrovirus, EBV-associated lymphoproliferation, hemophagocytic lymphohistiocytosis, or second cancers associated with CD19 CAR-T cells were reported. EQ-5D scores revealed a decrease from baseline in patient-reported health-related quality of life at 4 weeks, but improvements in mobility, independence, daily activities, and overall health status (EQ-5D visual analog scale) were seen by 3 months, and by 6 months, most patients had returned to baseline or better overall health status (Table 7).

[0268] TIFF0007813701000016.tif116163 * The EQ-5D visual analogue scale (VAS) assesses overall health status on a scale of 0 to 100, with higher scores indicating better health status. EQ-5D, European Quality of Life-5 Dimensions; N / A, not applicable; SD, standard deviation.

[0269] Sixteen patients (24%) who received CD19 CAR-T cells died (n=14 [21%]), primarily due to PD. Two patients had grade 5 AEs (3%), including one patient with organizing pneumonia related to conditioning chemotherapy and one patient with staphylococcal bacteremia related to conditioning chemotherapy and CD19 CAR-T cell therapy.

[0270] The median time to peak anti-CD19 CAR T-cell levels was 15 days (range, 8-31) after CD19 CAR-T-cell infusion, and in the presence of normal median B-cell levels, cells were still detectable at 24 months in some patients (6 / 10 [60%]) with evaluable samples at the time of data cutoff. Circulating CAR T-cell persistence over time, as measured by qPCR, showed a decline over time in patients who continued to respond and in those who relapsed.

[0271] The rapid proliferation, return to baseline, and clearance over time are consistent with the known mechanism of action of anti-CD19 CAR T cells bearing CD28 and CD3ζ costimulatory domains. All four patients who did not respond to CD19 CAR-T cell therapy had detectable B cells at baseline and no B-cell aplasia was observed at any time point during the study. Although no association with baseline tumor burden was observed, proliferation was associated with response (P=0.0036), with area under the curve (AUC) and peak being >200-fold higher in responders versus nonresponders, and with similar trends in MRD-negative versus MRD-positive patients at week 4. For both CRS and NE, proliferation was higher in patients with grade ≥3 events versus grade ≤2 events, with the highest peak and AUC observed in patients receiving tocilizumab ± steroids after CD19 CAR-T cell infusion. The median time to peak for assessed cytokines was 8 days, with most recovering to baseline levels by day 28. Serum granulocyte-macrophage colony-stimulating factor and interleukin (IL)-6 were associated with grade ≥3 CRS and NE. Serum ferritin was associated only with grade ≥3 CRS, whereas serum IL-2 and interferon-γ were associated only with grade ≥3 NE. Furthermore, cerebrospinal fluid cytokine analysis revealed higher levels of C-reactive protein, ferritin, IL-6, IL-8, and vascular cell adhesion molecule 1 in patients with grade ≥3 NE. Induction of anti-CAR antibodies was not observed in any patients.

[0272] The rates of grade ≥3 CRS and NE were similar to those previously reported with anti-CD19 CAR T-cell therapy in aggressive NHL (Neelapu SS et al. The New England Journal of Medicine 2017;377:2531, Schuster SJ et al. The New England Journal of Medicine 2019;380:45). There were no deaths due to CRS or NE, and most symptoms developed early during treatment, were generally reversible, and were not associated with long-term clinical sequelae that impaired activities of daily living. The observed association between peak serum cytokines and grade ≥3 CRS and / or grade ≥3 neurological events, which corresponded to the rise and peak levels of CAR T cells in the blood, indicates a role for CD19 CAR-T cells in these toxicities. The association between toxicities and peak levels of CAR and myeloid cell-associated serum cytokines, chemokines, and effector molecules is consistent with previously published data using similar CAR constructs in NHL. 10、13 Although one case of grade 4 cerebral edema occurred, the patient fully recovered and remains in complete response at 24 months of follow-up, with no unresolved neurological sequelae. Patient-reported outcomes also suggest no long-term quality of life impairment after CD19 CAR-T cell therapy. Example 3

[0273] This example provides additional analysis for the above study. Eligible patients (≥18 years old) with R / R MCL had an ECOG score of 0-1 and had received ≤5 prior therapies, including chemotherapy, anti-CD20 antibodies, and BTKi. Patients received leukapheresis and conditioning chemotherapy (cyclophosphamide 300 mg / m² / d and fludarabine 30 mg / m² / d for 3 days on days -5, -4, and -3), followed by a single IV infusion of 2 x 10 6Patients received a single infusion of CD19 CAR-T at a target dose of CAR T cells / kg. The CD19 CAR construct contains the CD3ζ T cell activation domain and the CD28 signaling domain. The manufacturing process removed circulating CD19-expressing leukemia cells from the leukapheresis product. Sabatino M, et al. Blood 2016;128:1227.

[0274] Some patients received both bridging regimens consisting of dexamethasone (20-40 mg or equivalent PO or IV, once daily for 1-4 days), ibrutinib (560 mg PO, once daily), or acalabrutinib (100 mg PO, twice daily) administered after leukapheresis. These regimens were completed within 5 days before the start of conditioning chemotherapy and required PET-CT scans after bridging. The primary endpoint was objective response rate (ORR [complete response (CR) + partial response]). Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), OS, frequency of adverse events (AEs), blood CAR T cell levels, and serum cytokine levels. Efficacy and safety analyses included all patients who received CD19 CAR T-cell therapy.

[0275] Key inclusion criteria were: R / R MCL, defined as disease progression after the most recent regimen or failure to achieve a CR or PR to the most recent regimen; 1 to 5 prior therapies, which must have included anthracycline- or bendamustine-containing chemotherapy, an anti-CD20 monoclonal antibody, and ibrutinib or acalabrutinib; 1 or more measurable lesions; age 18 years or older; ECOG score of 0 or 1; and adequate bone marrow, renal, hepatic, pulmonary, and cardiac function. Key exclusion criteria were: prior autologous stem cell transplantation (allo-SCT); prior CD19-targeted therapy; prior CAR-T cell therapy; clinically relevant infection; and previous or current CNS involvement by MCL or other CNS disorders.

[0276] A total of 68 patients received CD19 CAR-T cell therapy. Presented herein are updated safety (68 patients) and efficacy (60 patients) results, with a median follow-up of 12.3 months (range 7.0-32.3). A total of 28 patients (47%) had a follow-up period of ≥24 months. The median time to initial response was 1.0 month (range 0.8-3.1), and the median time to complete response was 3.0 months (range 0.9-9.3). Of the 24 patients (40%) who converted from PR / SD to CR, 21 (35%) converted from PR to CR, and 3 patients (5%) converted from SD to CR.

[0277] The median age was 65 years (range, 38-79), and 39 patients (57%) were male. One hundred percent of patients had an ECOG score of 0 / 1, 25% had blastoid morphology, 85% had stage IV disease, 56% had intermediate / high-risk MIPI, 81% had received three or more prior lines of therapy (median 3 lines, range, 1-5), 99% had received a prior anthracycline or bendamustine, 100% had received a prior anti-CD20 monoclonal antibody, and 100% had received a prior BTK therapy (ibrutinib 85%, acalabrutinib 24%, and both 9%). Forty-three patients (43%) relapsed after autologous SCT; 56% were refractory to ibrutinib and 12% were refractory to acalabrutinib. In 34 / 49 patients with available data, the Ki-67 index was ≥ 50%. Twenty-five (37%) patients received bridging therapy (ibrutinib 21%, acalabrutinib 7%, dexamethasone 18%, methylprednisolone 3%, both BTKis and steroids 9%, ibrutinib and steroids 6%, acalabrutinib and steroids 3%), and 23 / 25 patients underwent PET-CT after bridging to confirm measurable disease before CD19 CAR-T cell infusion (20 / 23 had a mean SPD of ≥ 50 mm from screening). 2 3 / 23 people had SPD mm from screening 2 had decreased slightly).

[0278] High ORRs were observed in both efficacy-evaluable and intention-to-treat (ITT) patients. 95% achieved a concordant ORR, and 90% achieved a concordant CR. The investigator-assessed ORR in 60 efficacy-evaluable patients was 88% (95% CI, 77%-95%), with a CR rate of 70% (95% CI, 57%-81%) and a PR rate of 18% (95% CI, 10%-30%). The IRRC-assessed ORR in 60 efficacy-evaluable patients was 93% (95% CI, 84%-98%), with a CR rate of 67% (95% CI, 53%-78%) and a PR rate of 27% (95% CI, 16%-40%). ORRs were consistent across key subgroups (age, MCL morphology, Ki-67 index, disease stage, abbreviated MIPI, steroid use for AE management, tocilizumab use, and bridging therapy use). The investigator-assessed ORR in ITT patients was 80% (95% CI, 69%-88%), with a CR rate of 59% (95% CI, 47%-71%) and a PR rate of 20% (95% CI, 12%-31%).The IRRC-assessed ORR in ITT patients was 85% (95% CI, 75%-92%), with a CR rate of 59% (95% CI, 47%-71%) and a PR rate of 26% (95% CI, 16%-37%).

[0279] The median DOR was not reached after a median follow-up of 12.3 months. Fifty-seven percent of all patients and 78% of CR patients remained in remission. The first 28 treated patients had a median follow-up of 27.0 months (range, 25.3-32.3), of which 43% remained in remission without further treatment. The median PFS and OS were not reached after a median follow-up of 12.3 months. The 12-month PFS rate (95% CI) was 61% (45%-74%). The 12-month OS rate (95% CI) was 83% (71%-91%).

[0280] More than 35% of patients experienced treatment-emergent adverse events (grade 1, 0%; grade 2, 1%; grade 3, 16%; grade 4, 76%; and grade 5, 3%). The most common grade ≥3 AEs (occurring in ≥20% of patients) were neutropenia (69%, grade 4), thrombocytopenia (35%, grade 4), anemia (50%, grade 3), and hypophosphatemia (22%, grade 3). There were no patient deaths due to cytokine release syndrome (CRS). Grade ≥3 CRS, as assessed by Lee DW, et al. Blood. 2014, 124:188, was reported in 15% of patients. The most common symptoms of CRS of any grade were hypotension (51%), hypoxia (34%), and fever (91%). Adverse event management included tocilizumab (59%) and corticosteroids (22%). The median time to onset was 2 days (range 1–13), the median duration was 11 days, and 62 / 62 (100%) patients with CRS of any grade had resolved events.

[0281] Neurologic events (NE) of any grade were reported in 63% of patients (31% had grade ≥3 NE) and included encephalopathy (31%), confusional state (21%), and tremor (35%). No patients died from neurologic events. One patient had grade 4 cerebral edema that fully resolved with aggressive multimodality treatment, including ventriculostomy and IV rabbit antithymocyte globulin (ATG). All CRS events and most NE (37 / 43 patients) were reversible. The median time to onset and duration of NE were 7 days (range, 1-32) and 12 days, respectively.

[0282] Higher peak CAR T cell levels were associated with responders (objective response) rather than nonresponders. Higher peak CAR T cell levels were associated with negative rather than positive MRD at week 4. The median time to peak anti-CD19 CAR T cell levels after CD19 CAR T cell infusion was 15 days (range, 8-31). Anti-CD19 CAR T cells were detectable at 24 months in most patients with evaluable samples (6 / 10 [60%]). Expansion was associated with response and MRD status. Expansion was greater in patients with CRS of grade ≥3 versus CRS of grade ≤2 and neurological events.

[0283] Several associations were observed between peak serum biomarker levels and toxicity. Measures associated with grade ≥3 CRS included IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and perforin. Measures associated with grade ≥3 neurological events included IL-2, IL-1Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. Measures associated with both grade ≥3 CRS and neurological events included IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B.

[0284] The CD19 CAR-T cell therapy described herein, administered as a single infusion, demonstrated a high rate of durable responses in R / R MCL. The 93% ORR, including a 67% CR rate, represents the highest reported rate of disease control in patients whose prior BTK therapy failed. Of the first 28 patients treated, 43% remained in remission after 24 months or more of follow-up. The safety profile was consistent with that reported in previous studies of anti-CD19 CAR T-cell therapy in aggressive NHL: there were no deaths due to CRS or neurological events, and most symptoms developed early in treatment and were generally reversible. The efficacy, reliability, rapid manufacturing, and manageable toxicity highlight the role of the CD19 CAR-T cell therapy described herein in treating patients with R / R MCL, a disease with unmet medical need. Example 4

[0285] This example provides additional analysis for the clinical trial described above. Eligible patients were aged 18 years or older, had pathologically confirmed MCL with evidence of either cyclin D1 overexpression or the presence of t(11;14), and had relapsed / refractory disease to one to five prior regimens for MCL. Prior therapy must have included anthracycline- or bendamustine-containing chemotherapy, an anti-CD20 monoclonal antibody, and ibrutinib or acalabrutinib. All patients received prior BTK therapy. Patients must have received prior BTK therapy, but it did not have to be their most recent therapy prior to study entry, and patients did not need to be resistant to BTK therapy. Eligible patients had an absolute lymphocyte count ≥ 100 / μL. Patients who underwent autologous SCT within 6 weeks of CD19 CAR-T infusion or previous CD19-targeted therapy or allogeneic SCT were excluded. All patients underwent leukapheresis to obtain cells for manufacturing of CD19 CAR-T cell therapy. Patients received optional bridging therapy, including dexamethasone (20-40 mg or equivalent PO or IV daily for 1-4 days), ibrutinib (560 mg orally (PO) daily), or acalabrutinib (100 mg PO twice daily). The manufacturing process involved CD4+ / CD8 + This product is a modification of axicabtagene ciloleucel to eliminate circulating lymphoma cells by positive enrichment of the cells. This product is referred to herein as "CAR T cells." This product may also be identified as KTE-X19. Fludarabine (30 mg / m 2 / day) and cyclophosphamide (500 mg / m 2 / day) on days -5, -4, and -3, followed by 2 x 10 6 A single intravenous infusion of CD19 CAR-T cells was administered at CAR T cells / kg. More details regarding patient treatment can be found in Example 2.

[0286] The study had two goals. First, to compare the pharmacological profiles of CAR T products in low-risk and high-risk patients in the ZUMA-2 clinical trial, as defined by TP53 (tumor protein p53) gene mutation status and Ki-67 tumor proliferation index. Patients with high-risk MCL characteristics, including tumor protein p53 gene (TP53) mutations and a high Ki-67 proliferation index, typically have a poor prognosis with current standard therapy (Cheah CY, et al. J Clin Oncol. 2016;34:1256-1269). Low-risk patients in this analysis had a Ki-67 proliferation index of <50% (by central review) or wild-type TP53, while high-risk patients had a Ki-67 of ≥50% or a TP53 mutation by next-generation sequencing. In the primary efficacy analysis of ZUMA-2 (N=60), the ORR was 93% (67% CR) after a median follow-up of 12.3 months. Responses continued in 57% of all patients and 78% of patients with a CR. ORRs were generally comparable between low-risk and high-risk patients in ZUMA-2, including those with a Ki-67 proliferation index < or ≥ 50% and unmutated vs. mutated TP53. Wang M, et al. New Engl J Med. 2020;382:1331-1342.

[0287] A secondary goal was to confirm the pharmacodynamic profile in patients who achieved minimal residual disease (MRD)-negativity early (day 28) and in patients with grade 4 neurotoxicity. In a previous analysis of ZUMA-2 results, blood CAR T cell levels by peak and area under the curve (AUC) from days 0 to 28 were associated with ORR (including undetectable MRD) and grade ≥3 CRS and neurological events. Wang M, et al. New Engl J Med. 2020;382:1331-1342. In that analysis, CRS and neurological events were primarily reversible (N=68 treated patients), with 15% having grade ≥3 CRS, 31% having grade ≥3 neurological events, and 2 having grade 5 AEs (one of which was CAR T product-related). MRD (sensitivity 10 -5 ) was assessed by next-generation sequencing as previously reported. Wang M, et al. New Engl J Med. 2020;382:1331-1342.

[0288] In this update, we report pharmacology data for all 68 treated patients in ZUMA-2, in which CAR T-cell product characteristics, blood CAR T-cell levels, serum cytokine levels, and their associations with clinical outcomes were analyzed using previously described methods. Locke FL, et al. Mol Ther. 2017;25:285-295. Wilcoxon rank-sum tests were used to measure associations between subgroup outcomes and CAR T-cell and cytokine levels. P values ​​were not corrected for multiple testing.

[0289] CAR T-cell product characteristics were generally comparable across prognostic groups defined by Ki-67 proliferation index and TP53 mutation status, with a trend toward a more differentiated phenotype in the high Ki-67 subgroup and a CD4-based phenotype in TP53-mutated patients (Table 8).

[0290] TIFF0007813701000017.tif60163 aOf all 68 treated patients, product characterization data were available for 65 total patients. Product characterization data were available for 48 / 49 total patients for whom Ki-67 data were available, and for all 36 patients for whom TP53 mutation data were available. TP53, tumor protein p53 gene

[0291] Equivalent CAR T-cell proliferation was observed in groups with different prognostic factors defined by Ki-67 proliferation index and TP53 mutation status. Both the peak levels and AUC of CAR T cells in the blood after administration were comparable in patients with wild-type versus mutant TP53 or Ki-67 proliferation index <50% versus ≥50%, consistent with comparable efficacy in these subgroups. The primary endpoint, objective response rate (ORR) in patients, is shown in Table 9. The median time to response was 28 days (range: 24-92 days), with a median follow-up time of 12.3 months. Twenty-eight patients had potential follow-up of 24 months or more, and 12 of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

[0292] The ORR was 100% vs. 94% in patients with a Ki-67 proliferation index <50% vs. ≥50%, and the CR rate was 64% vs. 78% in patients with a Ki-67 proliferation index <50% vs. ≥50%. Table 9. The number of patients with available data for the Ki-67 proliferation index was 49.

[0293] TIFF0007813701000018.tif23128

[0294] The ORR was 100% in both patients with wild-type versus mutant TP53, and the CR rate was 67% versus 100% for wild-type versus mutant TP53. Table 10. The number of patients with available data for TP53 was 36. All six patients with TP53 mutations and all 30 patients without mutations responded. Of the six patients with TP53 mutations, three had grade ≥3 neurotoxicity and two had grade ≥3 CRS.

[0295] TIFF0007813701000019.tif23128

[0296] Up to 44 biomarkers in serum, including IL (interleukin); INF-γ (interferon gamma), MCP-1 (monocyte chemoattractant protein-1), IL-2Rα (IL-2 receptor α), sPD-L1 (soluble programmed death-ligand 1), and sVCAM (soluble vascular cell adhesion molecule), were measured before treatment, on day 0, and at various time points up to day 28 after CAR T-cell infusion. The pharmacodynamic profiles of the two prognostic groups with a Ki-67 proliferation index <50% versus ≥50% were comparable for proliferative (IL-15, IL-2), inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1), immunoregulatory (IFN-γ, IL-10), chemokines (IL-8 and MCP-1), and effector cytokines (granzyme B). Furthermore, there was a trend toward increased levels of proliferative (IL-15, IL-2) and proinflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1) cytokines in patients with mutant versus wild-type TP53 (Figures 1A-1F).

[0297] There was also a trend toward increased peak levels of selected cytokines in serum in patients who achieved MRD-negative status. MRD was analyzed in 29 of 68 patients (43%). Of these patients, 24 (83% [19 patients with a complete response and 5 with a partial response]) were MRD-negative at 1 month after CAR T-cell administration. One month after CAR T-cell administration, MRD-negative patients (n=24 / 29) had increased median peak levels of interferon (IFN)-γ and interleukin (IL)-6 compared with MRD-positive patients (n=5 / 29), and there was a trend toward increased IL-2. Cytokine levels peaked in serum within 7 days of treatment. Consistent trends were also observed for PD-L1 and granzyme B. Increased peak CAR T-cell levels measured within 14 days after treatment were also observed in patients who were MRD-negative at 1 month (Figures 2A-2I).

[0298] Six patients developed grade 4 neurological events, including one cerebral edema. Three patients had concurrent grade 4 CRS. Patients with grade 4 neurological events had increased peak levels of proinflammatory serum biomarkers (e.g., IFNγ, MCP-1, TNF-α, IL-2, and IL-6) compared with patients without neurological events.

[0299] Cerebral edema resolved completely after aggressive multidisciplinary treatment. Wang M, et al. New Engl J Med. 2020;382:1331-1342. This patient had the highest CAR T cell proliferation and peak serum IL-2 levels, and elevations of multiple cytokines were several-fold higher in this patient compared with median values ​​in other studies / ZUMA-2 patients. Table 11.

[0300] TIFF0007813701000020.tif90163 a Of the 66 patients with available data.

[0301] The pharmacokinetic and pharmacodynamic profiles of CAR T cells were comparable across MCL patient groups, but different prognostic marker status (defined by Ki-67 and mutant TP53) was associated with lower and higher risk, consistent with comparable clinical response rates. Patients with mutant TP53 showed a trend toward higher levels of proinflammatory markers.

[0302] The pharmacodynamic profile of CAR T-cell administration was associated with efficacy (MRD status at month 1) and grade 4 treatment-emergent neurological events. Patients who developed cerebral edema had the highest peak CAR T-cell levels and serum IL-2, as well as elevated pro-inflammatory markers after treatment. Example 5

[0303] A phase 2, single-arm clinical trial was conducted evaluating CD19-directed, genetically modified autologous T-cell immunotherapy for the treatment of patients with relapsed or refractory mantle cell lymphoma (MCL) who had received at least one prior therapy, which may have included anti-CD20 antibodies, anthracycline- or bendamustine-containing chemotherapy, and / or Bruton's tyrosine kinase inhibitors (BTKi), such as ibrutinib or acalabrutinib. Eligible patients also had disease progression after previous therapy and disease resistant to their most recent therapy. The study excluded patients with active or severe infection, prior allogeneic hematopoietic stem cell transplantation (HSCT), detectable cerebrospinal fluid malignant cells or brain metastases, and any history of central nervous system (CNS) lymphoma or CNS disorder.

[0304] Patient peripheral blood mononuclear cells were obtained by leukapheresis. The mononuclear cells were enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-deficient viral vector containing FMC63-28Z CAR, a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable fragment (scFv), CD28, and CD3ζ domains. Without being bound by any hypothesis, selection for CD4+ and CD8+ cells may reduce the likelihood of circulating CD19-expressing tumor cells in the patient's leukapheresis material during the ex vivo manufacturing process. The T cell product of this process may be identified as KTE-X19. The anti-CD19 CAR T cells were expanded, washed, formulated into a suspension, and cryopreserved. Prior to receiving anti-CD19 CAR T-cell therapy, patients received intravenous cyclophosphamide 500 mg / m on each of the following days: 5, 4, and 3 days prior to CAR T-cell infusion. 2 and intravenous fludarabine 30 mg / m 2 Patients were treated with a lymphodepleting chemotherapy regimen of 100 mg / kg / day. Patients may also have received acetaminophen and diphenhydramine or another H1 antihistamine approximately 30–60 minutes before anti-CD19 CAR T-cell infusion. Prophylactic use of systemic corticosteroids was avoided because they may interfere with CAR T-cell activity.

[0305] The target dose is 2 × 10 per kg of body weight. 6 CAR-positive viable T cells or anti-CD19 CAR T cells, up to 2 x 10 8 anti-CD19 CAR T cells (for patients weighing 100 kg or more). Sixty-eight patients received a single infusion of anti-CD19 CAR T cells (via either gravity or peristaltic pump over approximately 30 minutes), and 60 of these patients were followed for at least 6 months after disease assessment at week 4 to qualify them as efficacy-evaluable. Fifty-six patients received 2 x 10 6 One patient received 1 × 10 anti-CD19 CAR T cells / kg. 6 Dose of anti-CD19 CAR T cells / kg, 1 patient was 1.6 × 10 6 dose of anti-CD19 CAR T cells / kg, 1.8 × 10 in two patients; 6 dose of anti-CD19 CAR T cells / kg, and -2 patients 1.9 × 10 6Anti-CD19 CAR T cells were administered at a dose of 1000 mg / kg. Of these 60 patients, the median age was 65 years (range: 38-79 years), 51 were male, and 56 were Caucasian. Fifty patients had stage IV disease. Based on the simplified Mantle Cell Lymphoma International Prognostic Index (s-MIPI), 25 patients were classified as low-risk, 25 as intermediate-risk, 8 as high-risk, and 2 as unknown-risk. Twenty patients underwent baseline bone marrow biopsy performed per protocol; of these, 10 were negative, 8 were positive, and 2 were indeterminate. The median number of prior lines of therapy for all 60 efficacy-evaluable patients was 3 (range: 2-5). Twenty-six patients had relapsed after or were refractory to autologous HSCT. Twenty-one patients had relapsed after their most recent treatment for MCL, and 36 patients were refractory to their most recent treatment for MCL. Fourteen patients had blastoid MCL. Following leukapheresis and prior to anti-CD19 CAR T cell infusion, 21 patients received bridging therapy: 19 patients were treated with a BTKi, 14 patients were treated with corticosteroids, and 6 patients were treated with both a BTKi and corticosteroids. Fifty-three patients received intravenous cyclophosphamide 500 mg / m , both administered 5, 4, and 3 days prior to anti-CD19 CAR T therapy (day 0). 2 and intravenous fludarabine 30 mg / m 2 Seven patients received the same dose of lymphodepleting chemotherapy for at least four days before CART therapy. The primary endpoint, the objective response rate (ORR) in patients, is shown in Table 12. The median time to response was 28 days (range: 24-92 days), with a median follow-up time of 12.3 months. Twenty-eight patients had potential follow-up of at least 24 months, and 12 of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

[0306] TIFF0007813701000021.tif90163CI, confidence interval; NE, not estimable; NR, not reached; PR, partial response. Among all responders. DOR is measured from the date of first objective response to the date of progression or death. b.Censored value. Cytokine release syndrome (CRS) was observed in 75 of 82 patients, including CRS of grade 3 or greater (Lee grading system 1) in 15 of 82 patients. The median time to CRS onset was 3 days (range, 1-13), and the median duration of CRS was 10 days (range, 1-50). Among patients with CRS, the major symptoms (i.e., symptoms occurring in >10% of patients...

Claims

1. 1. A composition for treating mantle cell lymphoma (MCL) in a subject in need thereof, comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR), the composition comprising: The anti-CD19 CAR comprises an anti-CD19 single-chain variable fragment (scFv) containing heavy and light chain variable regions of FMC63, a CD28 intracellular signaling region, and a CD3ζ signaling domain. composition.

2. 2. The composition of claim 1, wherein the MCL is relapsed or refractory (R / R) MCL.

3. The composition described in claim 2, wherein the MCL is classical, blastoid, or pleomorphic MCL.

4. 4. The composition of any of claims 1 to 3, wherein the MCL is resistant to or has relapsed after one or more of chemotherapy, radiation therapy, immunotherapy, autologous stem cell transplantation, or any combination thereof.

5. 10. The composition of claim 1, wherein the subject is resistant to or has relapsed after one or more of autologous SCT, chemotherapy including anti-CD20 antibodies, anthracyclines or bendamustine, and / or a Bruton's tyrosine kinase inhibitor (BTKi).

6. 6. The composition of claim 5, wherein the BTKi is ibrutinib or acalabrutinib.

7. the subject undergoes bridging therapy for MCL after leukapheresis and before lymphodepleting chemotherapy; the bridging therapy is selected from treatment with dexamethasone, methylprednisolone, ibrutinib, and / or acalabrutinib; immunomodulatory agents; R-CHOP, bendamustine; alkylating agents; and / or platinum-based agents, and the bridging therapy is completed within 5 days; 2. The composition of claim 1, wherein the lymphocyte-depleting chemotherapy is intravenous cyclophosphamide 500 mg / m2 and intravenous fludarabine 30 mg / m2, both of which are administered 5 days, 4 days, and 3 days before drug infusion, respectively.

8. 2. The composition of claim 1, wherein the autologous T cells comprise CD4+ and CD8+ CAR T cells prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment for T cells prior to transduction of the polynucleotide encoding the CAR.

9. 9. The composition of claim 8, wherein the PBMCs are enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-deficient viral vector containing the polynucleotide.

10. 9. The composition of claim 8, wherein the autologous T cells contain fewer cancer cells than T cells from a leukapheresis product that has not been positively selected for CD4+ and CD8+ T cells.

11. 9. The composition of claim 8, wherein the autologous T cells comprise naive-like T cells rather than T cells derived from a leukapheresis product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

12. 10. The composition of claim 9, wherein the autologous T cells have a lower proportion of differentiated T cells, an increased proportion of CD3+ cells, reduced IFN-γ production, and / or a lower proportion of CD3- cells than T cells from a leukapheresis product that has not been positively selected / enriched for CD4+ and CD8+ T cells.

13. The subjects with MCL had a mean blood glucose level of 1.8 x 10 per kg of body weight. ６ , 1.9 × 10 ６ , or 2 x 10 ６ CAR-positive viable T cells, up to 2 x 10 ８ 9. The composition of claim 8, wherein the CAR-positive viable T cells (for a patient weighing 100 kg or more) are administered one or more times.

14. 9. The composition of claim 8, wherein if the subject achieves a complete response to the first infusion, the subject may receive a second infusion of anti-CD19 CAR T cells, and if the subject progresses after more than three months of remission, provided that CD19 expression is maintained and neutralizing antibodies to the CAR are not suspected, and response is assessed using the Lugano classification.

15. 9. The composition of claim 8, wherein after T cell administration, the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity.

16. 16. The composition of claim 15, wherein the subject is monitored for signs and symptoms of CRS and neurotoxicity for at least 7 days or daily for 4 weeks after injection.

17. 16. The composition of claim 15, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and the signs or symptoms associated with a neurological event include encephalopathy, convulsions, altered level of consciousness, speech disturbance, tremor, and confusion.

18. 16. The composition of claim 15, wherein CRS in a subject with MCL is managed according to the following protocol:

19. 16. The composition of claim 15, wherein neurotoxicity in a subject with MCL is managed according to the following protocol:

20. 9. The composition of claim 8, wherein the subject with MCL is a high-risk patient as determined by a Ki-67 tumor proliferation index of 50% or greater and / or the presence of a TP53 mutation.

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