Influence of tumor microenvironment on curative effect of immunotherapy

Abstract

The present disclosure relates to the prognosis and impact of tumor microenvironment on the efficacy of cell-based immunotherapies (e.g., T cell therapy, TCR-based therapy, and CAR-based therapy).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 490,870, filed March 17, 2023; U.S. Provisional Patent Application No. 63 / 491,516, filed March 21, 2023; U.S. Provisional Patent Application No. 63 / 496,887, filed April 18, 2023; and U.S. Provisional Patent Application No. 63 / 502,295, filed May 15, 2023, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to methods for diagnosing and prognosing subjects receiving immunotherapy, compositions for immunotherapy, and immunotherapy using these compositions. Background Technology

[0004] Axi-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy initially approved for the treatment of relapsed / refractory (R / R) large B-cell lymphoma (LBCL) in adults who have received ≥2 lines of systemic therapy. ZUMA-7 (NCT03391466) was the first randomized, global, multicenter phase 3 study to compare axi-cel with historical standard of care (SOC) as second-line therapy in R / R LBCL patients. Standard of care consisted of two or three cycles of investigator-selected platinum-based chemotherapy, designed to allow chemotherapy-sensitive patients to subsequently receive high-dose chemotherapy combined with autologous stem cell transplantation (HDT-ASCT). Axi-cel demonstrated superior efficacy compared to standard of care with manageable safety. In the primary analysis, the hazard ratio (HR) for event-free survival (EFS) was 0.398 (P < 0.0001; median EFS was 8.3 months in the axi-cel group and 2.0 months in the SOC group, with estimated 24-month EFS rates of 40.5% and 16.3%, respectively). Despite these promising results, a significant number of patients still exhibit primary resistance (no response) or secondary resistance (relapse after initial response) to CAR-T cell therapy, thus requiring further investigation into potential biomarkers associated with treatment resistance.

[0005] In LBCL, known prognostic factors include high tumor burden, lactate dehydrogenase (LDH) elevation, activated B-cell (ABC)-like molecular subtype, age, and systemic inflammatory markers such as interleukin-6 (IL-6) and C-reactive protein (CRP) based on clinical and real-world evidence from the era of chemotherapy. In the era of cell therapies, tumor burden and LDH were negatively associated with efficacy of CAR T cell therapy as shown in ZUMA-1 (third-line or higher line LBCL). In addition, the quality and quantity of pre-treatment tumor T cell infiltration, as characterized by ImmunoSign 21 (IS21; T cell gene expression signature) and Immunoscore (immunohistochemistry [IHC] using CD3 cells and CD8 cells), were positively associated with outcomes of CAR T cell therapy. Conversion data from real-world patients treated with CAR T cell therapy further underscore the impact of associations between tumor-associated chronic inflammation, checkpoint ligand upregulation, myeloid cell inhibition of CAR T cell function, and patterns of tumor genomic complexity with CAR T cell outcomes. However, predictive biomarkers for CAR T cell intervention across lines of therapy have not been definitively established, and the association between tumor gene expression profiles and response to CAR T cells has not been exhaustively studied. Furthermore, while the predictive and prognostic role of the tumor microenvironment (TME) in solid tumors has been well described, the importance of intratumoral immune context to CAR T cell therapy remains unclear.

[0006] To meet these needs, we analyzed pre-treatment tumor characteristics in the ZUMA-7 study to discover tumor-specific features that can predict efficacy of axi-cel or SOC. SUMMARY

[0007] It should be understood that the application of the present disclosure is not limited to the details of the following embodiments, claims, descriptions, and drawings. The present disclosure can have other embodiments and can be practiced or carried out in various other ways.

[0008] Provided herein are methods relating to the assessment of certain parameters, e.g., expression of certain biomarkers or analytes, that can be correlated with outcomes, such as treatment outcomes, including responses, such as complete response (CR) or partial response (PR); or safety outcomes (e.g., adverse events), such as toxicity occurrence, e.g., neurotoxicity or cytokine release syndrome (CRS), that occur following administration of an immunotherapy, such as a cell therapy. Also provided are methods for assessing the likelihood of a response and / or the likelihood of a toxicity risk based on the assessment of a parameter, such as expression of a biomarker or analyte in a patient. Also provided are methods and uses for treating a subject having a disease or condition, typically or including a cancer or tumor, such as a leukemia or lymphoma, with an immunotherapy (e.g., a T cell, non-T cell, TCR-based therapy, CAR-based therapy, bispecific T cell engager (BiTE), and / or immune checkpoint blockade), including a cell (e.g., an engineered T cell) and / or a composition thereof. In some aspects, the methods and uses provide or achieve an improved response and / or a more durable response or efficacy and / or a reduced risk of toxicity or other side effects in a subject treated with some methods, as compared to certain alternative methods. In some embodiments, the methods include administration of a specified number or relative number of engineered cells, administration of a determined ratio of certain types of cells, treatment of a particular patient population (such as those having a particular risk profile, stage, and / or prior treatment history), administration of an additional therapeutic agent, and / or combinations thereof.

[0009] In one aspect, the present disclosure relates to immunotherapy products. As a non-limiting example, one aspect of the present disclosure relates to the use of Yescarta as a second-line therapy. In certain aspects, without being bound by any particular theory, the primary overall survival (OS) analysis results from the Phase 3 ZUMA-7 study demonstrated that Yescarta showed a statistically significant improvement in OS compared to historical treatment; the historical treatment was the standard of care (SOC) for relapsed / refractory large B-cell lymphoma (R / R LBCL) patients who relapsed within 12 months of completing first-line therapy in the context of curative treatment over nearly 30 years. This is a multi-step process that includes platinum salvage combination chemoimmunotherapy regimens followed by high-dose therapy (HDT) and stem cell transplant (ASCT) for patients who respond to salvage chemotherapy. OS was designated as a clinically important pre-specified key secondary endpoint, which was defined as the length of time from randomization to death from any cause.

[0010] In one aspect, the present disclosure provides a method of predicting the likelihood of a patient in need thereof responding to a cell therapy product, comprising the steps of: quantitatively detecting the gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; and determining the likelihood of the patient responding to the cell therapy product based at least in part on the gene expression level. In such an embodiment, an elevated gene expression level of at least one gene, as compared to a control value, indicates an increased likelihood of response or a decreased likelihood of response as compared to a predetermined response rate likelihood, the gene expression level being quantitatively detected from a patient sample, and the patient sample being collected from the patient prior to treatment with the cell therapy product.

[0011] In one aspect, the present disclosure provides a method for treating a malignant tumor in a patient, comprising the steps of: quantitatively detecting a gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; determining, based at least in part on the quantitatively detecting the gene expression level of the at least one gene, whether an effective dose of a cell therapy product should be administered to the patient as a second line therapy or as a third line therapy; and administering the effective dose of the cell therapy product as a second line therapy or as a third line therapy based on the determining step. In certain embodiments, if the gene expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A reaches or is higher than a control value of the at least one gene, then an effective dose of the cell therapy product is administered to the patient as a second line therapy; or if the gene expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is lower than the control value of the at least one gene, then an effective dose of the cell therapy product is administered to the patient as a third line therapy; or if the gene expression level of at least one of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 reaches or is lower than a control value of the at least one gene, then an effective dose of the cell therapy product is administered to the patient as a second line therapy; or if the gene expression level of at least one of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 reaches or is higher than a control value of the at least one gene, then an effective dose of the cell therapy product is administered to the patient as a third line therapy.

[0012] In one embodiment, the immunotherapy is a T cell therapy. In some embodiments, the T cell therapy comprises an adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. In one particular embodiment, the eACT comprises administration of engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, the eACT comprises administration of engineered antigen-specific T cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is a CAR T cell or TCR T cell therapy. In one embodiment, the immunotherapy is an anti-CD19 CAR T cell therapy.

[0013] The following are non-limiting embodiments of the present disclosure.

[0014] In at least a first aspect, the present disclosure provides a method of predicting the likelihood of a patient in need thereof having a response to a cell therapy product, comprising the steps of:

[0015] quantitatively detecting a gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; and

[0016] determining the likelihood of the patient having a response to the cell therapy product based at least in part on the gene expression level,

[0017] wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of a response or a decreased likelihood of a response compared to a predetermined likelihood of a response rate, and

[0018] wherein the gene expression level is quantitatively detected from a patient sample, and the patient sample is collected from the patient prior to treatment with the cell therapy product.

[0019] In certain aspects, the at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of a response compared to a predetermined likelihood of a response rate.

[0020] In certain aspects, the at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of response compared to a predetermined likelihood of response.

[0021] In certain aspects, the at least one gene exhibits an at least 20% elevated CD19 expression level relative to a CD19 control expression level, an at least 40% elevated MS4A1 expression level relative to a MS4A1 control expression level, and an at least 60% elevated TNFRSF17 expression level relative to a TNFRSF17 control expression level, indicating an increased likelihood of response compared to a predetermined likelihood of response.

[0022] In certain aspects, the at least one gene is selected from the group consisting of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates a decreased likelihood of response compared to a predetermined likelihood of response.

[0023] In certain aspects, response is defined as one or more of complete response, partial response, durable response, progression-free survival, or event-free survival.

[0024] In certain aspects, the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

[0025] In certain aspects, the cell therapy product is autologous or allogeneic.

[0026] In certain aspects, the target antigen is a tumor antigen, preferably selected from tumor-associated surface antigens such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD79a, CD79b, CD123, FLT3, BCMA, SLAMF7, CD8, CLL-1, c-Met, CMV-specific antigens, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal-epithelial mucin, EBV-specific antigens, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight-melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11 R alpha, IL-13R-a2, influenza virus-specific antigens;CD38, insulin growth factor (IGF1)-1, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage- specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 (PCTA-1), prostate-specific membrane antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stroma antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), GPC3 (glypican-3), and any derivative or variant of these antigens.

[0027] In certain aspects, the cell therapy product expresses a chimeric antigen receptor comprising a CD28 costimulatory domain.

[0028] In certain aspects, the patient has been diagnosed with a cancer / tumor selected from the group consisting of a solid tumor, a sarcoma, a carcinoma, a lymphoma, a multiple myeloma, Hodgkin’s disease, non-Hodgkin’s lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (not specified type), follicular lymphoma (FL), DLBCL arising from FL, transformed follicular lymphoma, high grade B-cell lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), one or more of acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom’s macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and Oki disease), head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, hepatocellular cancer, prostate cancer, breast cancer, or a combination thereof.

[0029] In certain aspects, the cancer is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) not specified type, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

[0030] In certain aspects, the cell therapy product is selected from axicinumab, brexucabtagene autoleucel, tisagenlecleucel, lisocabtagene maraleucel, and bb2121.

[0031] In certain aspects, the cell therapy product is administered as a second line therapy.

[0032] In certain aspects, the patient sample is a tumor biopsy.

[0033] In certain aspects, the present disclosure provides a method for treating a malignant tumor in a patient, comprising the steps of:

[0034] quantitatively detecting a gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2;

[0035] determining, based at least in part on the quantitatively detecting the gene expression level of the at least one gene, whether an effective dose of the cell therapy product should be administered to the patient as a second line therapy or as a third line therapy; and

[0036] administering the effective dose of the cell therapy product as a second line therapy or as a third line therapy based on the determining step,

[0037] wherein the gene expression level is quantitatively detected from a patient sample, and the patient sample is collected from the patient prior to treatment with the cell therapy product,

[0038] wherein if the gene expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is at or above a control value for the at least one gene, the patient is administered an effective dose of the cell therapy product as a second line therapy, or

[0039] wherein if the gene expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is at or below a control value for the at least one gene, the patient is administered an effective dose of the cell therapy product as a third line therapy, or

[0040] wherein if the gene expression level of at least one of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or below a control value for the at least one gene, the patient is administered an effective dose of the cell therapy product as a second line therapy, or

[0041] wherein if the gene expression level of at least one of the genes of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or above a control value for the at least one gene, the patient is administered an effective dose of a cell therapy product as a third line therapy.

[0042] In certain aspects, the gene expression level of at least one of the genes of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is below a control value for the at least one gene, or if the gene expression level of at least one of the genes of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or above a control value for the at least one gene, the patient is administered a second line treatment regimen for a malignancy that does not include a cell therapy.

[0043] In certain aspects, the at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A.

[0044] In certain aspects, the at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17.

[0045] In certain aspects, the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

[0046] In certain aspects, the cell therapy product is autologous or allogeneic.

[0047] In certain aspects, the target antigen is a tumor antigen, preferably selected from tumor-associated surface antigens such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD79a, CD79b, CD123, FLT3, BCMA, SLAMF7, CD8, CLL-1, c-Met, CMV-specific antigens, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal-epithelial mucin, EBV-specific antigens, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight-melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11 R alpha, IL-13R-a2, influenza virus-specific antigens;CD38, insulin growth factor (IGF1)-1, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage- specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 (PCTA-1), prostate-specific membrane antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stroma antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), GPC3 (glypican-3), and any derivative or variant of these antigens.

[0048] In certain aspects, the cell therapy product expresses a chimeric antigen receptor comprising a CD28 costimulatory domain.

[0049] In certain aspects, the patient has been diagnosed with a cancer / tumor selected from the group consisting of a solid tumor, a sarcoma, a carcinoma, a lymphoma, a multiple myeloma, Hodgkin’s disease, non-Hodgkin’s lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (not specified type), follicular lymphoma (FL), DLBCL arising from FL, transformed follicular lymphoma, high grade B-cell lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), one or more of acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom’s macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and Oki disease), head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, hepatocellular cancer, prostate cancer, breast cancer, or a combination thereof.

[0050] In certain aspects, the cancer is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) not specified type, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

[0051] In certain aspects, the cell therapy product is selected from the group consisting of axicinumab, belantumab, tisagenlecleucel, liso-cel, and bb2121.

[0052] In certain aspects, the patient sample is a tumor biopsy.

[0053] In certain aspects, a method of treating a subject having a lymphoma is disclosed, comprising the steps of: administering to the subject a debulking regimen; and administering to the subject an immunotherapy after the debulking regimen.

[0054] In certain aspects, the debulking regimen comprises any of the options contained in Table 39. In certain aspects, the debulking regimen comprises at least 2 of the options contained in Table 39.

[0055] In certain aspects, the immunotherapy comprises anti-CD19 CAR T cells.

[0056] In certain aspects, a debulking regimen is administered to a subject when the subject has a tumor burden above a predetermined level. In certain aspects, a debulking regimen is not administered to a subject when the subject has a tumor burden below a predetermined level.

[0057] In one aspect, the present disclosure relates to a method of predicting the likelihood of a patient in need thereof responding to a cell therapy product, comprising the steps of: quantitatively detecting the gene expression level of at least two genes selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A; calculating a composite expression score, the calculation comprising adding the gene expression levels of the at least two genes; and determining the likelihood of the patient responding to the cell therapy product based at least in part on the composite expression score. In such an embodiment, an increase in the composite expression score compared to a control value indicates an increased likelihood of response compared to a predetermined likelihood of response rate. In such an embodiment, the composite expression score is calculated as follows. The individual expression levels of each of the at least two genes from CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A are measured. These individual expression levels are then normalized and averaged to generate a composite expression score for the at least two genes. The generated composite expression score is then compared to a control value, wherein the control value is the historical median of historical composite expression scores for the at least two genes from other patients.

[0058] In one aspect, the present disclosure relates to a method of predicting the likelihood of a patient in need thereof responding to a cell therapy product, comprising the steps of: quantitatively detecting the gene expression level of at least two genes selected from the group consisting of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; calculating a composite expression score, the calculation comprising adding the gene expression levels of the at least two genes; and determining the likelihood of the patient responding to the cell therapy product based at least in part on the composite expression score. In one such embodiment, an increase in the composite expression score compared to a control value indicates a decreased likelihood of response compared to a predetermined likelihood of response rate. In one such embodiment, the composite expression score is calculated as follows. The individual expression levels of each of at least two genes from BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 are measured. These individual expression levels are then normalized and averaged to generate a composite expression score for the at least two genes. The generated composite expression score is then compared to a control value, wherein the control value is the historical median of historical composite expression scores for the at least two genes from other patients.

[0059] In one aspect, the present disclosure relates to a method for treating a malignant tumor in a patient, comprising the steps of: quantitatively detecting the gene expression level of at least two genes selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A; calculating a composite expression score, which calculation comprises adding the gene expression levels of the at least two genes; and determining, at least in part based on the composite expression score, whether an effective dose of a cell therapy product should be administered to the patient as a second line therapy or an effective dose of a cell therapy product should be administered to the patient as a third line therapy; and based on the determining step, administering an effective dose of a cell therapy product as a second line therapy or as a third line therapy. In one such embodiment, if the composite expression score of at least two of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A reaches or is above a control value, then an effective dose of a cell therapy product is administered to the patient as a second line therapy, or if the composite expression score of at least two of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A reaches or is below a control value, then an effective dose of a cell therapy product is administered to the patient as a third line therapy. In one such embodiment, the composite expression score is calculated as follows. The individual expression level of each of the at least two genes from CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is measured. These individual expression levels are then normalized and averaged to generate a composite expression score for the at least two genes. The generated composite expression score is then compared to a control value, wherein the control value is the historical median of historical composite expression scores of the at least two genes from other patients.

[0060] In one aspect, the present disclosure relates to a method for treating a malignant tumor in a patient, comprising the steps of: quantitatively detecting the gene expression level of at least two genes selected from the group consisting of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; calculating a composite expression score, which calculation comprises adding the gene expression levels of the at least two genes; and determining, at least in part based on the composite expression score, whether an effective dose of a cell therapy product should be administered to the patient as a second line therapy or an effective dose of a cell therapy product should be administered to the patient as a third line therapy; and based on the determining step, administering an effective dose of a cell therapy product as a second line therapy or as a third line therapy. In one such embodiment, if the composite expression score of at least two of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 reaches or falls below a control value, then an effective dose of a cell therapy product is administered to the patient as a second line therapy, or if the composite expression score of at least two of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 reaches or rises above a control value, then an effective dose of a cell therapy product is administered to the patient as a third line therapy. In one such embodiment, the composite expression score is calculated as follows. The individual expression level of each of the at least two genes from BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is measured. These individual expression levels are then normalized and averaged to generate a composite expression score for the at least two genes. The generated composite expression score is then compared to a control value, wherein the control value is the historical median of historical composite expression scores of the at least two genes from other patients. DETAILED DESCRIPTION

[0061] The present disclosure is based, in part, on the discovery that pre-treatment gene expression levels of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A are positively correlated with an increased likelihood of responding to a cell therapy, while pre-treatment gene expression levels of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 are negatively correlated with a likelihood of responding to a cell therapy. This discovery informs whether a patient should be administered a cell therapy.

[0062] In one aspect, the present disclosure relates to immunotherapy products. As a non-limiting example, one aspect of the present disclosure relates to the use of Yescarta as a second line therapy. In certain aspects, without being bound by any particular theory, the primary overall survival (OS) analysis results from the Phase 3 ZUMA-7 study demonstrated that Yescarta showed a statistically significant improvement in OS compared to historical treatment; the historical treatment was the standard of care (SOC) for relapsed / refractory large B-cell lymphoma (R / R LBCL) patients who relapsed within 12 months after completing first-line therapy in the context of curative treatment over nearly 30 years. This is a multi-step process that includes platinum salvage combination chemoimmunotherapy regimens followed by high-dose therapy (HDT) and stem cell transplant (ASCT) for patients who respond to salvage chemotherapy. OS was designated as a clinically important pre-specified key secondary endpoint, which was defined as the length of time from randomization to death from any cause.

[0063] Definitions

[0064] To facilitate a better understanding of the present disclosure, certain terms are defined first below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0065] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0066] As used herein, the term “or” is understood to encompass both “or” and “and,” unless the context clearly dictates otherwise.

[0067] The term "and / or" as used herein is to be taken as specific disclosure of each of the various components of the home or components in the home that, individually or in combination, can be claimed as inventive. Thus, as used herein, the term "and / or" encompasses the various components individually or in combination, for purposes of the disclosure and the appended claims. Likewise, the term "and / or" as used herein encompasses the individual provisos as well as in combination, for purposes of the disclosure and the appended claims.

[0068] The terms "for example," "e.g.," and "for instance," as used herein, are to be understood as non-limiting terms of illustration, not as a limitation on the remainder of the disclosure.

[0069] The terms "or more," "at least," "exceeding," and the like, such as "at least one," are understood to include but not be limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated value. Any greater number or fraction in between is also included.

[0070] In contrast, the term“no more than” includes every value less than the stated value. For example,“no more than 100 nucleotides” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 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.

[0071] The terms“a plurality,”“at least two,”“two or more,”“at least a second,” and the like, are understood to include, without limitation, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

[0072] Throughout this specification, the word “comprising” or variations such as “including” or “having” shall be understood to imply inclusion of the stated elements, integers, or steps, or groups of elements, integers, or steps, but not to exclude any other elements, integers, or steps, or groups of elements, integers, or steps. It should be understood that wherever aspects are described using the language “comprising” herein, other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided. The term “consisting of” excludes any element, step, or ingredient not specified in the claims. Relating to Gray, 53 F.2d 520, 11 USPQ255 (CCPA 1931); and Davis, 80 USPQ 448, 450 (Bd.App.1948) (“consisting of” is defined as “closing a claim to include materials different from those described, except for impurities usually associated with them”). The term "consistent with essentially" limits the scope of the claim to the specified materials or steps and those materials or steps that do not substantially affect the basic and novel features of the claimed disclosure.

[0073] Unless otherwise specified or apparent from the context, as used herein, the term “about” refers to a value or composition within an acceptable margin of error 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, according to practice in the art, “about” or “approximately” may mean within one or more standard deviations. “About” or “approximately” may mean a range of up to 10% (i.e., ±10%). Thus, “about” can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or processes, these terms may mean up to one order of magnitude or up to five times a value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” shall be assumed to be within the acceptable margin of error for that particular value or composition.

[0074] As stated herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, and, where appropriate, its fraction (such as one-tenth and one-hundredth of an integer), unless otherwise stated.

[0075] The units, prefixes, and symbols used in this article are provided in their SI-accepted form. Numerical ranges include the numbers that define the range.

[0076] Unless defined otherwise, 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 is related. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2nd ed. CRC Press, 2001; "The Dictionary of Cell & Molecular Biology," 5th ed. Academic Press, 2013; and "The Oxford Dictionary Of Biochemistry And Molecular Biology," Cammack et al. eds., 2nd ed. Oxford University Press, 2006, provide one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0077] "Administering" means physically introducing a pharmaceutical agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). Exemplary routes of administration for the compositions disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). As used herein, the phrase "parenteral administration" means modes of administering 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, and in vivo electroporation. In some embodiments, the formulation is administered by a non-parenteral route (e.g., orally). Other non-parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In one embodiment, CAR T cell therapy is administered via an "infusion product" comprising CAR T cells.

[0078] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen. In general, an antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0079] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, univalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above antibodies. In some embodiments, an antibody described herein refers to a polyclonal antibody population.

[0080] An "antigen binding molecule," "antigen binding moiety," or "antibody fragment" refers to any molecule comprising an antigen binding portion of an antibody from which the molecule is derived (e.g., CDRs). An antigen binding molecule can comprise 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. Peptibodies (i.e., Fc fusion molecules comprising a peptide binding domain) are another example of suitable antigen binding molecules. In some embodiments, an antigen binding molecule binds an antigen on a tumor cell. In some embodiments, an antigen binding molecule binds an antigen on a cell involved in a hyperproliferative disease or a viral or bacterial antigen. In some embodiments, an antigen binding molecule binds CD19. In further embodiments, an antigen binding molecule is an antibody fragment, including one or more complementarity determining regions (CDRs) thereof, that specifically binds an antigen. In further embodiments, an antigen binding molecule is a single chain variable region fragment (scFv). In some embodiments, an antigen binding molecule comprises or consists of an avimer.

[0081] An "antigen" refers to any molecule that elicits an immune response or that is capable of being bound by an antibody or antigen binding molecule. The immune response can involve antibody production, or activation of specific immune-competent cells, or both. Those skilled in the art will readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be expressed endogenously, i.e., from genomic DNA, or can be expressed recombinantly. An antigen can be specific to a certain tissue, such as a cancer cell, or it can be widely expressed. In addition, fragments of larger molecules can serve as antigens. In some embodiments, an antigen is a tumor antigen.

[0082] 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 effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof directly blocks a binding site on the ligand, or alters the binding ability of the ligand through indirect means (e.g., a structural or energetic change in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents a protein to which it binds from performing a biological function.

[0083] The term "autologous" refers to any material derived from the same individual and reintroduced into that individual at a later time. For example, the engineered autologous cell therapy (eACT ™ ) methods described herein involve collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and subsequently administering them back to the same patient.

[0084] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0085] In one embodiment, the CAR T cell therapy comprises "axicabtagene ciloleucel therapy." Axicabtagene ciloleucel therapy consists of a single infusion of anti-CD19 CAR transduced autologous T cells administered intravenously at a target dose of 2 x 10 6 anti-CD19 CAR T cells / kg. For subjects weighing more than 100 kg, a maximum flat dose of 2 x 10 8 anti-CD19 CAR T cells can be administered. The anti-CD19 CAR T cells are autologous human T cells that have been engineered to express an extracellular single-chain variable fragment (scFv) with specificity for CD19 linked to an intracellular signaling portion composed of signaling domains from CD28 and CD3 zeta (CD3-ζ) molecules arranged in tandem, the anti-CD19 CAR vector construct has been designed, optimized, and initially tested at the Surgical Branch of the National Cancer Institute (NCI, IND 13871) (Kochenderfer et al., J Immunother. 2009; 32(7):689-702; Kochenderfer et al., Blood. 2010; 116(19):3875-86). The scFv is derived from the variable region of the anti-CD19 monoclonal antibody FMC63 (Nicholson et al., Molecular Immunology. 1997; 34(16-17): 1157-65). A portion of the CD28 costimulatory molecule was added because murine models suggested that this was important for the antitumor effects and persistence of anti-CD19 CAR T cells (Kowolik et al., Cancer Res. 2006; 66(22): 10995-1004). The signaling domain of the CD3-ζ chain was used for T cell activation. These fragments were cloned into a murine stem cell virus (MSGV1)-based vector for genetic engineering of autologous T cells. The CAR construct was inserted into the genome of T cells by retroviral vector transduction. Briefly, peripheral blood mononuclear cells (PBMCs) were obtained by leukapheresis and Ficoll separation. The peripheral blood mononuclear cells were activated by culture with anti-CD3 antibody in the presence of recombinant interleukin 2 (IL-2). Stimulated cells were transduced with a retroviral vector containing the anti-CD19 CAR gene and propagated in culture to generate sufficient engineered T cells for administration. In some embodiments, the CAR T cell therapy is Yescarta ® ( axicabtagene ciloleucel). In some embodiments, the CAR T cell therapy is Tecartus® (Benotriptyline).

[0086] The terms "transduce" and "transduced" refer to the process of introducing exogenous DNA into a cell by a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr viral vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentivirus vector, or any combination thereof.

[0087] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors, which are able to invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include a tumor. In the present application, the term cancer is synonymous with malignant tumor. Examples of cancers that can be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods disclosed herein can be used to reduce the size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, multiple myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal B- cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) including non T-cell ALL, chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary gland tumor, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is NHL. A particular cancer can be responsive to chemotherapy or radiation therapy, or the cancer can be refractory. Refractory cancer refers to a cancer that is inapt for surgical intervention and which initially does not respond to chemotherapy or radiation therapy, or which becomes unresponsive over time.

[0088] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. Anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

[0089] As used herein, “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 to mediate a response in the second cell. As used herein, “cytokine” refers to a protein released by one cell that acts as an intercellular mediator on another cell. Cytokines can be expressed endogenously by a cell or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and proinflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-la, IL-lb, 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 cell adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0090] A “chemokine” is a cytokine that mediates chemotaxis or directed 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 chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus activation-regulated chemokine (TARC or CCL17).

[0091] As used herein, a "chimeric receptor" refers to an engineered surface-expressed molecule capable of recognizing a specific molecule. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) comprising a binding domain capable of interacting with a specific tumor antigen allow T cells to target and kill cancer cells expressing the specific tumor antigen. In one embodiment, T cell therapy is based on T cells engineered to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising (i) an antigen binding molecule, (ii) a costimulatory domain, and (iii) an activating domain. The costimulatory domain can comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a hinge domain that can be truncated.

[0092] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an engineered CAR T cell described in the specification, a small molecule, a "pharmaceutical agent") is any amount that, when used alone or in conjunction with another therapeutic agent, protects a subject against the onset of a disease or promotes the regression of a disease (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic disease periods, or the prevention of impairment or disability due to the disease). Such terms can be used interchangeably. The ability of a therapeutic agent to promote the regression of a disease can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during a clinical trial, in animal model systems predictive of efficacy in humans, or by assaying the activity of the pharmaceutical agent in an in vitro assay. Therapeutically effective amounts and dosing regimens can be determined empirically by testing in known in vitro or in vivo (e.g., animal model) systems.

[0093] The term "combination" refers to either a fixed combination in unit dosage form, or a combined administration where the compound of the disclosure and the combination partner (e.g., another drug as explained below, also referred to as a "therapeutic agent" or "pharmaceutical agent") can be administered independently at the same time or within a time interval, especially if these time intervals allow that the combination partners show a cooperative (e.g., synergistic) effect. The individual components can be packaged in a kit or packaged separately. One or both components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration. As used herein, the terms "co-administration" or "combined administration" and the like, are intended to encompass administration of the selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include treatment regimens in which the pharmaceutical agents are not necessarily administered by the same route of administration or at the same time.

[0094] The term "pharmaceutically acceptable" refers to a molecular entity or composition that is not harmful to its recipient, or any detrimental effects are outweighed by the beneficial effects to its recipient, when administered to a recipient. In the case of a vehicle, diluent, or excipient used in formulating a composition as disclosed herein, the pharmaceutically acceptable vehicle, diluent, or excipient must be compatible for its intended use with the other ingredients of the composition and not harmful to the recipient, or any detrimental effects must be outweighed by the beneficial effects to the recipient. The term "pharmaceutically acceptable vehicle" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, involved in carrying or transporting the active agent from one organ, or portion of the body, to another organ, or portion of the body. Each vehicle present in the pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or any detrimental effects must be outweighed by the beneficial effects to the recipient. Some examples of materials that can serve as pharmaceutically acceptable vehicles include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl

[0095] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable vehicles. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen showing a statistically significant probability of achieving a predetermined therapeutic effect when administered to an associated subject or population. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to forms suitable for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection, as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, such as an ointment, cream or controlled-release patch, or spray to the skin, lungs, or oral cavity; intravaginally or intrarectally, such as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and other mucosal surface administration.

[0096] The terms "reduce" and "reducing" are used interchangeably herein and indicate any change less than the original. "Reduce" and "reducing" are relative terms requiring a comparison between before and after a measurement. "Reduce" and "reducing" include complete depletion.

[0097] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or assayed substantially simultaneously with the test, measurement, or assay of interest. In some embodiments, the reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. When there is sufficient similarity to justify reliance on and / or comparison to the selected reference or control.

[0098] As used throughout, a "control value" refers to a historical value of a particular analyte observed in a population prior to administration of a cell therapy product. In some embodiments, a deviation from the historical value is associated with an increase or decrease in the likelihood of a particular patient responding to a cell therapy product in the patient's body relative to a predetermined and / or historical likelihood of responding to the cell therapy product. More specifically, in some embodiments, an elevated expression level of a certain analyte in a patient test sample relative to a control expression level of the corresponding analyte is associated with an increase in the chance of the patient responding to the cell therapy product in the patient's body relative to a predetermined and / or historical likelihood of responding to the cell therapy product. In certain embodiments, the increase in the chance of responding is measured relative to a known historical average likelihood of responding to the cell therapy product in a population. In some embodiments, an elevated expression level of a certain analyte in a patient test sample relative to a control expression level of the corresponding analyte is associated with a decrease in the chance of the patient responding to the cell therapy product in the patient's body relative to a predetermined and / or historical likelihood of responding to the cell therapy product. In certain embodiments, the decrease in the chance of responding is measured relative to a known and historical average likelihood of responding to the cell therapy product in a population.

[0099] As used herein, the term "predetermined" refers to an expected value or likelihood of an outcome based on information that does not include specific information related to any particular patient who can be or can become a subject of a cell therapy administration.

[0100] The terms“product” or“infusion product” are used interchangeably herein and refer to a T cell composition administered to a subject in need thereof. Typically, in CAR T cell therapy, the T cell composition is administered as an infusion product.

[0101] As used herein, the term“lymphocyte” includes a natural killer (NK) cell, a T cell, or a B cell. NK cells are a type of cytotoxic (cell-poisoning) lymphocyte that represents a major component of the innate immune system. NK cells reject tumors and cells infected by viruses. It acts through the process of apoptosis or programmed cell death. They are called“natural killers” because they do not need to be activated to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Its T cell receptor (TCR) distinguishes it from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell, or killer T cell), memory T cells ((i) stem memory TSCM cells (like naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but they also express large amounts of CD95, IL-2R beta, CXCR3, and LFA-1, and show many functional attributes characteristic of memory cells); (ii) central memory TCM cells express L-selectin and CCR7, they secrete IL-2 but not IFNgamma or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7, but produce effector cytokines (such as IFNgamma and IL-4)), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (with antibody involvement).

[0102] As is well known in the art, each type of T cell can be characterized by cell surface markers. For example, naive T cells can be characterized as CCR7+, CD45RO-, and CD95-. Additional markers for naive T cells include CD45RA+, CD62L+, CD27+, CD28+, CD127+, CD132+, CD25-, CD44-, and HLA-DR-. Surface markers for stem cell memory T cells (Tscm) include, but are not limited to, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, IL-7Ra+, CD95+, IL-2RP+, CXCR3+, and LFA-. Surface markers for effector memory T cells (Tem) include, but are not limited to, CCR7-, CD45RO+, and CD95+. Additional markers for effector memory T cells are IL-2R beta+. For central memory T cells (TCM), suitable markers include CD45RO+, CD95+, IL-2R beta+, CCR7+, and CD62L+. For effector T cells (Teff), suitable markers include, but are not limited to, CD45RA+, CD95+, IL-2R beta+, CCR7-, and CD62L-.

[0103] The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including but not limited to, deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), incorporated into the genome of the cell.

[0104] An "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 by any of these cells or the liver that selectively target, bind to, impair, destroy, and / or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells from the body of a vertebrate, or normal human cells or tissues in the context of autoimmunity or pathological inflammation.

[0105] The term "immunotherapy" refers to the treatment of a subject afflicted with, or at risk of, a disease or recurrence by a method involving inducing, enhancing, suppressing or otherwise altering an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™ ) and allogeneic T cell transplantation. However, one skilled in the art will recognize that the conditioning methods disclosed herein will enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 7,741,465, U.S. Patent No. 6,319,494, U.S. Patent No. 5,728,388, International Publication No. WO 2008 / 081035, International Publication No. WO 2015 / 20096, International Publication No. WO 2016 / 191756, International Publication No. WO 2016 / 191755, International Publication No. WO 2019 / 079564, and International Publication No. WO 2021 / 092290, each of which is incorporated herein in its entirety. In some embodiments, the immunotherapy comprises CAR T cell therapy. In some embodiments, the CAR T cell therapy product is administered via infusion.

[0106] The T cells of the immunotherapy can be from any source known in the art. For example, the T cells can be differentiated in vitro from a population of hematopoietic stem cells, or the T cells can be obtained from a subject. The 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 a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Additionally, the T cells can be derived from one or more T cell lines available in the art. The T cells can also be obtained from a blood unit collected from a subject using various techniques known to the skilled artisan, such as FICOLL ™ separation and / or apheresis. Additional methods for T cell isolation for T cell therapy, as well as methods for CAR T cell preparation for cell therapy, are disclosed in U.S. Patent Publication No. 2013 / 0287748, International Publication No. WO 2015 / 20096, International Publication No. WO 2016 / 191756, International Publication No. WO 2016 / 191755, International Publication No. WO 2019 / 079564, and International Publication No. WO 2021 / 092290, each of which is incorporated herein by reference in its entirety.

[0107] The term “engineered autologous cell therapy” or “eACT ™Adoptive cell transfer (also known as adoptive cell therapy) is a process by which a patient's own T cells are collected and then genetically engineered to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignancies. The T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen, which is linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activating domain. The CAR scFv can be designed to target, for example, CD 19, a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignancies, including but not limited to non-specified diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma, high grade B cell lymphoma, and DLBCL arising from follicular lymphoma, NHL, CLL, and non-T cell ALL. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, and these references are incorporated by reference in their entirety.

[0108] As used herein, "patient" or "subject" includes any human afflicted with a cancer (e.g., a lymphoma or a leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0109] As used herein, the term "ex vivo cell" refers to any cell cultured ex vivo. In particular, the ex vivo cell can include a T cell. The term "in vivo" refers to within a patient.

[0110] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues connected to each other by peptide bonds. A protein or peptide comprises at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, of which there are numerous types). "Polypeptide" includes, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural, recombinant, synthetic, or a combination thereof.

[0111] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell (e.g., T cell receptor (TCR) / CD3 complex) that specifically binds to a cognate stimulatory ligand presented on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when presented on an antigen presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) by the T cell. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies, MHC class I molecules loaded with peptides, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0112] As used herein, a "costimulatory signal" refers to a signal that, in conjunction with a primary signal such as TCR / CD3 ligation, results in a T cell response such as, but not limited to, proliferation and / or up- or down-regulation of key molecules.

[0113] 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, differentiation, etc. A costimulatory ligand induces a signal in addition to the primary signal provided by a stimulatory molecule, e.g., by binding of a T cell receptor (TCR) / CD3 complex to a major histocompatibility complex (MHC) molecule loaded with a peptide. Costimulatory ligands can include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds a 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. In certain embodiments, a costimulatory ligand includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule present on a T cell, 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).

[0114] A“costimulatory molecule” is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (a; b; d; e; g; z), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8a, CD8b, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc g receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, Ig a (CD79a), IL2R b, IL2R g, IL7R a, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0115] The terms "reduce" and "reducing" are used interchangeably herein and indicate any change less than the original. "Reduce" and "reducing" are relative terms requiring a comparison between before and after measurement. "Reduce" and "reducing" include complete ablation. Similarly, the term "increase" denotes any change above the original. "Increase," "higher," and "lower" are relative terms requiring a comparison between before and after measurement and / or between a reference standard. In some embodiments, the reference value is obtained from values of a general population, which can be a general patient population. In some embodiments, the reference value is from a quartile analysis of a general patient population.

[0116] "Treatment" of a subject refers to any type of intervention or process performed on the subject, or administration of an active agent to the subject, with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition associated with a disease or a biochemical indicator. In some embodiments, "treatment" includes partial alleviation. In another embodiment, "treatment" or "management" includes complete alleviation. In some embodiments, the treatment can be prophylactic, in which case the treatment is administered before any symptoms of the condition are observed. The term "prevention" as used herein refers to prophylactic or protective treatment of a disease or disease state. Prevention of a symptom, disease or disease state can include, for example, reduction (e.g., lessening) of one or more symptoms of the disease or disease state relative to a reference level (e.g., symptoms in a similar subject not administered the treatment). Prevention can also include delaying the onset of one or more symptoms of the disease or disease state, for example, relative to a reference level (e.g., onset of symptoms in a similar subject not administered the treatment). In embodiments, the disease is a disease described herein. In some embodiments, the disease is a cancer. In some embodiments, the disease state is CRS or neurotoxicity. In some embodiments, indicators of improvement or successful treatment include determining failure to exhibit a relevant score on a toxicity grading scale (e.g., CRS or neurotoxicity grading scale), such as a score of less than 3, or a change in grade or severity on a grading scale as discussed herein, such as a change from a score of 4 to a score of 3, or a change from a score of 4 to a score of 2, 1, or 0.

[0117] As used herein, "myeloid cells" are a subpopulation of white blood cells, including granulocytes, monocytes, macrophages, and dendritic cells.

[0118] In one embodiment, the terms "high" and "low" mean "above" and "below" the median of a representative subject population. In one embodiment, the terms mean the upper or lower quartile, respectively. Both mean and quartile distributions can be determined by one of ordinary skill in the art by routine methods.

[0119] As used herein, the term "quartile" is a statistical term describing the division of observations into four defined intervals based on the values of the data and how they compare to the entire set of observations.

[0120] As used herein, the term "study day 0" is defined as the day the subject receives the first infusion of CAR T cells. The day before study day 0 will be study day -1. Any day after enrollment and before study day -1 will be consecutive and a negative integer value.

[0121] As used herein, the term "durable response" refers to subjects who are in a continuous response for at least one year of follow-up after CAR T cell infusion. In one embodiment, "duration of response" is defined as the time from first objective response to disease progression or to death due to disease relapse.

[0122] As used herein, the term "relapse" refers to subjects who achieve a complete response (CR) or partial response (PR) and subsequently experience disease progression.

[0123] As used herein, the term "non-responder" refers to subjects who never experience a CR or PR after CAR T cell infusion, including subjects with stable disease (SD) and progressive disease (PD).

[0124] As used herein, the term "objective response" refers to complete response (CR), partial response (PR), or non-responder. Its assessment can be made according to the revised response evaluation criteria in malignant lymphoma international working group (Cheson et al., J Clin Oncol. 2007; 25(5):579-86).

[0125] As used herein, the term "complete response" refers to complete disappearance of disease, which becomes undetectable by radiological imaging and clinical laboratory assessments. There is no evidence of cancer at a given time.

[0126] As used herein, the term "partial response" refers to a tumor reduction of more than 30% but not complete disappearance.

[0127] As used herein, "objective response rate" (ORR) is determined according to the International Working Group (IWG) 2007 criteria (Cheson et al., J Clin Oncol. 2007; 25(5):579-86).

[0128] As used herein, "progression-free survival (PFS)" can be defined as the time from the date of T cell infusion to the date of disease progression or death from any cause. Progression is defined according to the response assessment results by the investigator following the IWG criteria (Cheson et al., J Clin Oncol. 2007; 25(5):579-86).

[0129] The term "overall survival (OS)" can be defined as the time from the date of T cell infusion to the date of death from any cause.

[0130] As used herein, expansion and persistence of CAR T cells in peripheral blood can be monitored by qPCR analysis, for example using CAR-specific primers directed to the scFv portion of the CAR (e.g., the heavy chain of the CD19 binding domain) and its hinge / CD28 transmembrane domain. Alternatively, it can be measured by calculating the number of CAR cells per unit blood volume.

[0131] As used herein, scheduled blood draws for CAR T cells are at pre-CAR T cell infusion, day 7, week 2 (day 14), week 4 (day 28), month 3 (day 90), month 6 (day 180), month 12 (day 360), and month 24 (day 720).

[0132] As used herein, "peak of CAR T cells" is defined as the maximum absolute number of CAR+ PBMC / µL reached in serum after day 0.

[0133] As used herein, "time to peak of CAR T cells" is defined as the number of days from day 0 to the day that the peak of CAR T cells is reached.

[0134] As used herein, "area under the curve (AUC) of CAR T cell levels from day 0 to day 28" is defined as the area under the curve in a plot of CAR T cell levels against scheduled visits from day 0 to day 28. This AUC measures the total level of CAR T cells over time.

[0135] As used herein, scheduled blood draws for cytokines are at pre-conditioning chemotherapy or the day of (day -5), day 0, day 1, day 3, day 5, day 7, every other day to hospitalization (if any), week 2 (day 14), and week 4 (day 28).

[0136] As used herein, "baseline" for cytokines is defined as the last value measured prior to conditioning chemotherapy.

[0137] As used herein, fold change from baseline on day X is defined as

[0138]

[0139] As used herein, "peak of cytokines post baseline" is defined as the maximum level of cytokines in serum reached up to day 28 after baseline (day -5).

[0140] As used herein, the“time to peak cytokine” following CAR T cell infusion is defined as the number of days from Day 0 to the day that the peak cytokine is reached.

[0141] As used herein, the“area under the curve (AUC) of cytokine levels” from Day -5 to Day 28 is defined as the area under the curve in a plot of cytokine levels versus scheduled visits from Day -5 to Day 28. This AUC measures the total level of cytokine over time. Given that cytokines and CAR+ T cells are measured at certain discrete time points, the trapezoidal rule can be used to estimate the AUC.

[0142] As used herein, a treatment-emergent adverse event (TEAE) is defined as an adverse event (AE) that occurs on or after the conditioning day of the first dose. Adverse events can be coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 22.0 and graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. Grading of cytokine release syndrome (CRS) events can be per the grading criteria for the syndrome established by Lee et al. (Lee et al., 2014 Blood. 2014; 124(2):188-95). Individual CRS symptoms can be graded according to CTCAE 4.03. Neurological events can be identified using a search strategy based on known neurological toxicities associated with CAR T immunotherapies, as described in, for example, Topp, MS, et al., Lancet Oncology. 2015; 16(1):57-66.

[0143] Various aspects of the present disclosure are described in further detail in the following subsections.

[0144] Characterization of serum protein profiles of immunotherapy cancer patients

[0145] In some embodiments, the present disclosure provides methods of characterizing the serum proteomic profile of a cancer patient prior to treatment with an immunotherapy and / or pre-conditioning. In one embodiment, the immunotherapy is selected from a chimeric receptor therapy (e.g., YESCARTA ™ axi-cel, TECARTUS ™ - brexucabtagene vicalt / KTE-X19, KYMRIAH ™(e.g., axicabtagene ciloleucel (Yescarta®), brexucabtagene celuloeucel (Tecartus®), etc.), TCR, TIL, immune checkpoint inhibitors, etc. In one embodiment, the immunotherapy product comprises autologous or allogeneic CAR T cells. In one embodiment, the immunotherapy comprises T cells modified with a T cell receptor. In one embodiment, the immunotherapy comprises tumor infiltrating lymphocytes (TILs). In one embodiment, the immunotherapy product comprises induced pluripotent stem cells (iPSCs). As described herein, in some embodiments, serum protein signatures are obtained by a pre-specified set of proteins and analyzed by OPIs and machine learning models. In some embodiments, serum levels can be measured by ELISA. In some embodiments, the serum protein profiles are associated with adverse events of chimeric receptor therapy (e.g., axicabtagene ciloleucel (axi-cel)) and can be used to predict adverse events in response to all immunotherapies (e.g., T cell, non-T cell, TCR-based therapies, CAR-based therapies, bispecific T cell engagers (BiTe), and / or immune checkpoint blockade).

[0146] In one embodiment, the present disclosure provides that baseline (pre-conditioning) serum levels of certain proteins associated with metabolic processes and leukocyte activation are positively correlated with adverse prognostic factors of immunotherapy, including international prognostic index and baseline tumor burden, and can be biomarkers thereof. In one embodiment, the immunotherapy is a T cell therapy. In some embodiments, the T cell therapy comprises an adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. In a particular embodiment, the eACT comprises administration of engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, the eACT comprises administration of engineered antigen-specific T cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is a CAR T cell or TCR T cell therapy. In one embodiment, the immunotherapy is an anti-CD19 CAR T cell therapy.

[0147] Accordingly, in one embodiment, the present disclosure provides a method of predicting international prognostic index and baseline tumor burden parameters in a cancer patient based on baseline (pre-conditioning) serum levels of metabolic process markers and / or leukocyte activation markers in the patient.

[0148] In one embodiment, the present disclosure indicates that an elevated expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A prior to treatment is associated with an increased likelihood of a patient responding to a cell therapy product in the patient, while an elevated expression level of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 prior to treatment is associated with a decreased likelihood of a patient responding to a cell therapy product in the patient. In one embodiment, this information is used to make decisions related to immunotherapy, including specifically: whether to administer immunotherapy, whether to administer immunotherapy as a second line therapy or a third line therapy, the dose of immunotherapy to administer, the dosing regimen to follow, and / or other agents to be administered to the patient prior to, after, and / or during administration of immunotherapy.

[0149] In one embodiment, a high level of a serum biomarker is at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold the level of the median. In one embodiment, a level of a protein biomarker is high or low, respectively, when the level of the protein biomarker falls to above or below the median or the above or below specified value by 0-0.1%, 0.1-0.5%, 0.5-1.0%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, etc., 95-100%. All listed values can be modified by the term "above."

[0150] In one embodiment, the present disclosure provides a method of treating a subject having a high tumor burden with an immunotherapy, wherein the high tumor burden is reduced by administering one or more agents or treatments that result in reduced inflammation (e.g., lower cytokine induction in the blood) and / or by using an alternative lymphodepletion regimen (that does not include administration of 500 mg / m 2 / day - 600 mg / m 2 / day - 600 mg / m 2 / day for 3 days) to reduce immune activation-mediated stress in the subject. In one embodiment, a subject has a high tumor burden (as assessed by SPD and / or tumor metabolic volume) when the baseline tumor burden (SPD) is greater than 2500 mm 2 , 3000 mm 2 , 3500 mm 2 , or 4000 mm 2 , preferably greater than 3000 mm 2 and / or the tumor metabolic volume is higher than the median of a representative tumor population (e.g., higher than 100 ml or higher than 150 ml).

[0151] In one embodiment, the disclosure provides a method of treating a subject having a high international prognostic index, wherein immune activation-mediated stress in the subject is reduced by administering one or more agents or treatments that result in reduced inflammation (e.g., lower cytokine induction in the blood) and / or by using an alternative lymphodepletion regimen prior to immunotherapy that does not include administering 500 mg / m 2 / day - 600 mg / m 2 / day of cyclophosphamide and 30 mg / m 2 / day of fludarabine for 3 days. In one embodiment, a subject has a high IPI when the international prognostic index (IPI) is greater than 1, 2, or 3.

[0152] In one embodiment, the immunotherapy is a T cell therapy. In one embodiment, the T cell therapy is autologous. In one embodiment, the T cell therapy is allogeneic. In some embodiments, the T cell therapy comprises an adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), iPSC, checkpoint inhibitors, and allogeneic T cell transplantation. In a particular embodiment, the eACT comprises administration of engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, the eACT comprises administration of engineered antigen-specific T cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is a CAR T cell or TCR T cell therapy. In one embodiment, the immunotherapy is an anti-CD19 CAR T cell therapy. Examples of target tumor antigens are listed elsewhere in the specification. Examples of cancers that can be treated by the methods of the disclosure are also provided elsewhere in the specification.

[0153] In one embodiment, the agent administered in combination with the immunotherapy and that reduces immune activation and / or endothelial cell disruption is selected from anti-IL-1 (e.g., anakinra), T cell activation inhibitor (e.g., dasatinib), JAK inhibitor (e.g., filgotinib), anti-GM-CSF (e.g., lenzilumab), anti-TNF (e.g., infliximab), Ang2 inhibitor (e.g., azilsartan), anti-angiogenic therapy (e.g., bevacizumab), anti-IFNg (e.g., ipilimumab), and the like, wherein the combination therapy reduces cytokine induction and / or wherein the combination therapy reduces endothelial cell disruption. In one embodiment, the immunotherapy is administered in a combination therapy that enhances T cell proliferation. In one embodiment, the combination therapy comprises treatment with pembrolizumab, lenalidomide, elotuzumab, and elotuzumab. In one embodiment, the therapy comprises monoralizumab (anti-CD47 antagonist), GSK3745417 (STING agonist), INCB001158 (ARG1 / 2 inhibitor), GS-1423 (CD73 x TGFp mAb), serplimab (CD40 agonist), GS3583 (FLT3 agonist), pecmetinib (CSF1R inhibitor), epacadostat (IDO1 inhibitor), GS9620 (TLR agonist).In one embodiment, the agent is selected from (i) a GM-CSF inhibitor selected from the group consisting of: lenzilumab; namilumab (AMG203); GSK3196165 / MOR103 / otlertimgab (GSK / MorphoSys); KB002 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022 / risdiplamab (Morphotek); or a biosimilar of any of them; E21R; and small molecules; (ii) a CSF1 inhibitor selected from the group consisting of: RG7155, PD-0360324, MCS110 / latrunculin A or a biosimilar version of any of them; and small molecules; and / or (iii) a GM-CSFR inhibitor and a CSF1R inhibitor selected from the group consisting of: Mavrilimumab (formerly CAM-3001; Medlmmune, Inc.); Cabiralimumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly and Company), emactuzumab, also known as RG7155 or RO5509554; FPA008 (Five Prime / BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041 / AFS98 / TG3003 (ElsaLys Bio, Transgene), SNDX-6352 (Syndax); a biosimilar version of any of them; and small molecules. In some embodiments, the additional therapy can be a cytokine (e.g., IL-2, IL-15), a stimulatory antibody (e.g., anti-41BB, OX-40), a checkpoint blockade (e.g., CTLA4, PD-1), or an innate immune stimulator (e.g., TLR, STING agonist). In some embodiments, the additional therapy used in combination can be a T-cell recruiting chemokine (e.g., CCL2, CCL1, CCL22, CCL17, and combinations thereof). In some embodiments, the additional therapy used in combination is administered systemically or intratumorally. In some embodiments, the additional therapy used in combination is administered with the conditioning and / or immunotherapy. In some embodiments, the additional therapy used in combination is administered sequentially with the conditioning and / or immunotherapy.

[0154] In an embodiment, an agent can / should be administered to the patient prior to, after and / or during the immunotherapy administration to reduce 3+ grade CRS in the subject. In an embodiment, the agent is administered to the patient prior to CAR-T infusion, prior to the peak of CAR-T expansion (e.g., day 0-6 post infusion) and / or at the time of peak CAR-T expansion (e.g., day 7-14). In an embodiment, the peak of CAR-T expansion is day 7-14 post infusion. In an embodiment, the peak of CAR-T expansion is day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19 or day 20 post infusion. In an embodiment, the period after peak CAR-T expansion is a period between day 14-28 post infusion. In an embodiment, the period after peak CAR-T expansion is day 1-day 5, day 5-day 10, day 10-day 15, day 15-day 20, day 20-day 25; any day after peak expansion day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 25, day 30, day 35, day 40, day 45, day 50.

[0155] In an embodiment, the immunotherapy is combined with low dose radiation, promotion of T cell activity and / or T cell agonists through immune checkpoint blockade. In an embodiment, the T cell agonist is selected from the group consisting of pembrolizumab, lenalidomide, an anti-CD40 antibody, and an anti-CD47 antibody. In an embodiment, the combination agent is selected from the group consisting of checkpoint inhibitors (e.g., anti-PD1 antibodies, pembrolizumab (Keytruda), cemiplimab (Libtayo), nivolumab (Opdivo); anti-PD-L1 antibodies, atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi); and / or anti-CTLA-4 antibodies, ipilimumab (Yervoy).

[0156] In an embodiment, the preconditioning regimen is a lymphodepletion regimen. In an embodiment, the lymphodepletion therapy regimen is selected from one of several possible regimens of cyclophosphamide / fludarabine, bendamustine, total body irradiation, anti-CD45 (epratuzumab), and other chemotherapeutic agents (e.g., AVM0703, busulfan, thiotepa / etoposide, pentostatin). Additional conditioning methods and regimens can be found elsewhere in this specification.

[0157] In one embodiment, the present disclosure provides a method of improving immunotherapy (e.g., CAR T cell therapy) by optimizing bridge therapy to modulate the tumor microenvironment to a more favorable immune permissive state. In one embodiment, the optimization comprises administering bridge therapy with an immunomodulatory imide drug (IMID) / cereblon modulator (e.g., lenalidomide, pomalidomide, ibrtinodide, and actimid). In one embodiment, the optimization comprises administering bridge therapy with local radiation.

[0158] In one embodiment, the present disclosure provides a method of improving immunotherapy (e.g., CAR T cell therapy) by optimizing bridge therapy to reduce tumor burden prior to administration of immunotherapy (e.g., CAR T cell therapy). In one embodiment, the optimization comprises administering bridge therapy with R-CHOP, bendamustine, an alkylating agent, and / or a platinum-based agent. Other exemplary bridge therapies are described elsewhere in the application.

[0159] In one embodiment, the present disclosure provides a method of improving immunotherapy (e.g., CAR T cell therapy) by optimizing conditioning therapy to modulate the tumor microenvironment to a more favorable immune permissive state (e.g., less myeloid inflammation in the TME). In one embodiment, the optimization comprises adding local radiation to cyclophosphamide / fludarabine conditioning. In one embodiment, the optimization comprises administering a platinum-based agent as a conditioning agent.

[0160] In one embodiment, the present disclosure provides a method of improving immunotherapy (e.g., CAR T cell therapy) by co-administering a biological response modifier or administering after immunotherapy (e.g., CAR T cell therapy) to enable CAR T cell activity. In one embodiment, the method comprises administering a gamma chain cytokine (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). In one embodiment, the method comprises administering a checkpoint blockade (e.g., anti-CTLA-4).

[0161] In one embodiment, the present disclosure provides a method of improving immunotherapy (e.g., CAR T cell therapy) by reprogramming T cells to overcome a deleterious tumor microenvironment, wherein the deleterious tumor microenvironment comprises a low T / M ratio, a high tumor burden, a high density of myeloid cells in the TME, and / or a high level of myeloid inflammation in the TME. In one embodiment, the T cells are engineered to express a gamma chain receptor cytokine. In one embodiment, the gamma chain receptor cytokine is expressed under a constitutive or inducible promoter.

[0162] In one embodiment, this disclosure provides a method for improving CAR T cell therapy by optimizing T cell production to help CAR T cells overcome a harmful tumor microenvironment, wherein a potentially harmful tumor microenvironment is characterized by a low T / M ratio, high tumor burden, high TME bone marrow cell density, and / or high TME bone marrow inflammation level. In one embodiment, a potentially harmful TME is characterized by a low T / M ratio (within -0.5 to 4), high tumor burden (within 3000 mmHg), and high tumor mass. 2 -40000mm 2 (internal), high bone marrow cell density (at 1000 cells / mm²) 2 -4000 cells / mm 2 (within) and / or high TME bone marrow inflammation levels (within 27-2000). In one embodiment, the method includes engineering CAR T cells to express γ-chain receptor cytokines. In one embodiment, the γ-chain receptor cytokines are expressed under a constitutive or inducible promoter. In one embodiment, the method includes growing T cells in the presence of γ-chain cytokines such as IL-15.

[0163] Clinical outcomes

[0164] In some implementations, the clinical outcome is a complete response. In some implementations, the clinical outcome is a sustained response. In some implementations, the clinical outcome is a complete response. In some implementations, the clinical outcome is no response. In some implementations, the clinical outcome is a partial response. In some implementations, the clinical outcome is an objective response. In some implementations, the clinical outcome is survival. In some implementations, the clinical outcome is relapse.

[0165] In some implementation schemes, the objective response (OR) is determined according to the revised IWG response criteria for malignant lymphomas (Cheson, 2007) and by means of the IWG response criteria for malignant lymphomas (Cheson et al., Journal of Clinical Oncology 32, Vol. 27 (September 2014), pp. 3059-3067). The duration of response is assessed. Investigator-assessed progression-free survival (PFS) is evaluated according to the Lugano response classification criteria.

[0166] In some embodiments, a portion of the clinical outcome is an assessment of adverse events. In this regard, CRS is graded according to Lee DW, et al. (2014). Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 Jul 10; 124(2): 188-195. Neurotoxicity is assessed by excluding other causes of neurological symptoms, by monitoring patients for signs and symptoms of neurotoxicity. Patients who develop > grade 2 neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. Intensive care support therapy is provided for severe or life- threatening neurotoxicity. In some embodiments, the symptoms of neurotoxicity are selected from the group consisting of encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

[0167] In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity at least daily for 7 days at an accredited medical facility following infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity for 4 weeks following infusion.

[0168] In some embodiments, the symptoms of neurotoxicity are selected from the group consisting of encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, the symptoms of adverse reactions are selected from the group consisting of 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), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, the patient is instructed to remain near the accredited medical facility for at least 4 weeks following infusion.

[0169] Clinical outcomes of CAR T cell therapy depend on the levels of CAR T cells in the blood. In some embodiments, the response, levels of CAR T cells in the blood, or immune-related factors are determined by follow-up at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the engineered CAR T cells. In some embodiments, the response, levels of CAR T cells in the blood, or immune-related factors are determined by follow-up at about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after administration of the engineered CAR T cells. In some embodiments, the response, levels of CAR T cells in the blood, and / or immune-related factors are determined by follow-up at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months after administration of the engineered CAR T cells. In some embodiments, the response, levels of CAR T cells in the blood, and / or immune-related factors are determined by follow-up at about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, or about 5 years after administration of the engineered CAR T cells.

[0170] In some embodiments, the methods described herein can provide clinical benefit to a subject. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients obtain clinical benefit. In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and any non-enumerated % therebetween of patients obtain clinical benefit. In some embodiments, the response rate is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, 10.5%, 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%, 25 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%, or 100%, or some other non-enumerated percentage, and some other non-enumerated range between 1% and 100%. In some embodiments, the response rate is between 0%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%. In some embodiments, the response rate is between 0%-1%, 1%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-15%, 15%-20%, 20-25%, 25%-30%, 35-40%, and so on up to 95%-100%.

[0171] Chimeric antigen receptors

[0172] In one embodiment, the immunotherapy is CAR-T cell immunotherapy. Chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be inserted into and expressed by immune cells, including T cells and other lymphocytes, according to techniques known in the art. Using CARs, a single receptor can be programmed to both recognize a specific antigen and, upon binding of that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens are present on tumor cells, the immune cells expressing CARs can target and kill the tumor cells. Chimeric antigen receptors can incorporate co-stimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0173] In some embodiments, the costimulatory domain comprising a truncated hinge domain (“THD”) further comprises some or all of an immunoglobulin family member, such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

[0174] In some embodiments, the THD is derived from a human complete hinge domain (“CHD”). In other embodiments, the THD is derived from a rodent, murine, or primate (e.g., non-human primate) CHD of the costimulatory protein. In some embodiments, the THD is derived from a chimeric CHD of the costimulatory protein.

[0175] The costimulatory domain of the CAR of the present disclosure can also comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain can be fused to the extracellular domain of the CAR. The costimulatory domain can similarly be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, this transmembrane domain is selected or modified by amino acid substitution to avoid its association with transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex. The transmembrane domain can be derived from natural or synthetic sources. Where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein.The transmembrane region used particularly in the present disclosure can be derived from (i.e., comprise) 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, Cytokine Receptor, DAP-10, DNAM1 (CD226), Fc gamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-Cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte Function-Associated Antigen 1 (LFA-1; CD11a / CD18), MHC Class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed Death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocyte Activation Molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF Receptor Protein, TNFR2, TNFSF14, Toll Ligand Receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncated form, or combination thereof.

[0176] Optionally, a short linker can form a connection between any or some of the extracellular, transmembrane, and intracellular domains of a CAR. The linker peptides described herein can also be used as a peptide tag. The linker peptide sequence can be of any suitable length to link one or more proteins of interest, and is preferably designed to be flexible enough to allow proper folding and / or function and / or activity of the one or two peptides it links. Thus, the linker peptide can be no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acids in length. In some embodiments, the linker peptide comprises a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids. In some embodiments, the linker comprises at least 7 and no more than 20 amino acids, at least 7 and no more than 19 amino acids, at least 7 and no more than 18 amino acids, at least 7 and no more than 17 amino acids, at least 7 and no more than 16 amino acids, at least 7 and no more than 15 amino acids, at least 7 and no more than 14 amino acids, at least 7 and no more than 13 amino acids, at least 7 and no more than 12 amino acids, or at least 7 and no more than 11 amino acids. In certain embodiments, the linker comprises 15-17 amino acids, and in particular embodiments, 16 amino acids. In some embodiments, the linker comprises 10 to 20 amino acids. In some embodiments, the linker comprises 14 to 19 amino acids. In some embodiments, the linker comprises 15 to 17 amino acids. In some embodiments, the linker comprises 15-16 amino acids. In some embodiments, the linker comprises 16 amino acids. In some embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0177] In some embodiments, a spacer domain is used. In some embodiments, the spacer domain is derived from CD4, CD8a, CD8b, CD28, CD28T, 4-1BB, or other molecules described herein. In some embodiments, the spacer domain can include a chemically induced dimerizer to control expression upon addition of a small molecule. In some embodiments, no spacer is used.

[0178] The intracellular (signaling) domains of the engineered T cells of the present disclosure can provide signaling to an activation domain, which then activates at least one normal effector function of the immune cell. The effector function of the T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0179] In certain embodiments, suitable intracellular signaling domains include (i.e., comprise), but are not limited to, 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, Cytokine Receptor, DAP-10, DNAM1 (CD226), Fc gamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-Cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, Lymphocyte Function-Associated Antigen 1 (LFA-1; CD11a / CD18), MHC Class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed Death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocyte Activation Molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF Receptor Protein, TNFR2, TNFSF14, Toll Ligand Receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncated forms, or combinations thereof.

[0180] Antigen binding molecules

[0181] Suitable CARs and TCRs can bind to an antigen, such as a cell surface antigen, by incorporation of an antigen binding molecule that interacts with the targeted antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, e.g., one or more single-chain antibody fragments (“scFv”). scFvs are single-chain antibody fragments having the variable regions of the heavy and light chains of an antibody joined together. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. scFvs retain the ability of the parent antibody to specifically interact with a target antigen. scFvs are useful in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain with other CAR components. Id. See also Krause et al., J. Exp. Med., vol. 188, no. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It will be appreciated that the antigen binding molecule is typically comprised within the extracellular portion of the CAR or TCR, such that it is able to recognize and bind to the antigen of interest. Bispecific and multispecific CARs and TCRs are contemplated within the scope of the present disclosure, which have specificity for more than one target of interest.

[0182] In some embodiments, the polynucleotide encodes a CAR or TCR comprising a (truncated) hinge domain and an antigen binding molecule that specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is selected from tumor-associated surface antigens such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigens, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal- epithelial mucin, EBV-specific antigens, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight-melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11R alpha, IL-13R-a2, influenza virus-specific antigens;CD38, insulin growth factor (IGF1)-1, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, muthsp70-2, mutated p53, mutated ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate carcinoma tumor antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stroma antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), and any derivative or variant of these surface antigens.

[0183] Engineered T cells and products

[0184] In one embodiment, the immunotherapy is a T cell therapy. In some embodiments, donor T cells for a T cell therapy are obtained from the patient (e.g., for an autologous T cell therapy). In other embodiments, donor T cells for a T cell therapy are obtained from a subject that is not the patient. In certain embodiments, the T cells are tumor infiltrating lymphocytes (TILs), engineered autologous T cells (eACT ™ ), allogeneic T cells, heterologous T cells, or any combination thereof. In some embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient afflicted with a cancer or a tumor. In some embodiments, the donor subject is a human patient not afflicted with a cancer or a tumor.

[0185] In one embodiment, the cells are obtained from a subject. In one embodiment, the cells are induced pluripotent stem cells (iPSCs). T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or differentiated in vitro. Additionally, T cells can be derived from one or more T cell lines available in the art. Various techniques known to the skilled artisan can also be used, such as FICOLL™ T cells are obtained from a unit of blood collected from a subject (e.g., by apheresis and / or leukapheresis). In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or media for subsequent processing. In some embodiments, cells are washed with PBS. It will be appreciated that a washing step can be used, such as by using a semi-automated flow-through centrifuge, e.g., Cobe™ 2991 Cell Processor, Baxter CytoMate® ™ In some embodiments, washed cells are resuspended in one or more biocompatible buffers or other salt solutions with or without buffers. In some embodiments, unwanted components of the apheresis sample are removed. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Publication 2013 / 0287748, which is incorporated by reference herein in its entirety.

[0186] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes (e.g., by using centrifugation through a PERCOLL ™ 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 T cell populations by negative selection can be accomplished using a combination of antibodies against surface markers specific for the cells being negatively selected. In some embodiments, cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry is used, which uses a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CDl lb, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to isolate the cell populations of interest for use in the present disclosure.

[0187] In some embodiments, PBMCs are used directly for genetic modification of immune cells (such as CARs) using methods as described herein. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations prior to or after genetic modification and / or expansion.

[0188] In some embodiments, the CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the phenotype markers of central memory T cells include expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and are negative for granzyme B. In some embodiments, the central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, the effector T cells are negative for CCR7, CD28, CD62L, and CD127 and are positive for granzyme B and perforin. In some embodiments, the CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

[0189] In some embodiments, the immune cells (e.g., T cells) are genetically modified (engineered) after isolation using known methods, or are activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification of the immune cells. In another embodiment, the immune cells (e.g., T cells) are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described by way of non-limiting example in U.S. Patent Nos. 6905874, 6867041, and 6797514, and International Publication Nos. WO 2015 / 20096, WO 2016 / 191756, WO 2016 / 191755, WO 2019 / 079564, and WO 2021 / 092290, each of which is incorporated by reference herein in its entirety. The contents of these patents are hereby incorporated by reference in their entirety. Generally, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents (such as anti-CD3 and anti-CD28 antibodies) typically attached to beads or other surfaces in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead act as a “surrogate” antigen presenting cell (APC). One example is Dynabeads ®System, a CD3 / CD28 activator / stimulator system for physiologic activation of human T cells. In other embodiments, T cells are activated and stimulated for proliferation with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patents 6,040,177 and 5,827,642 and PCT Publication WO 2012 / 129514 (the contents of these patents are hereby incorporated by reference in their entireties).

[0190] In some embodiments, the composition comprising engineered T cells comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient.

[0191] In some embodiments, the composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of those in the art. In some embodiments, the composition is maintained at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8, using a buffer. In some embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the composition described herein, with or without an additional therapeutic agent. In some embodiments, the vehicle for parenteral injection is sterile distilled water in which the composition described herein is formulated with or without at least one additional therapeutic agent into a sterile isotonic solution suitable for aseptic storage. In some embodiments, preparation involves formulating the desired molecule with a polymeric compound, such as polylactic or polyglycolic acid, a bead, or a liposome, to provide controlled or sustained release of the product, which is then delivered by depot injection. In some embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0192] In some embodiments, the engineered T cells are administered in a therapeutically effective amount. For example, a therapeutically effective amount of engineered 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 cells, or at least about 10 10 cells. In another embodiment, a therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 81010 cells. In some implementations, the therapeutically effective amount of T cells is approximately 2 × 1010. 6 cells / kg, approximately 3 × 10⁻⁶ 6 cells / kg, approximately 4 × 10⁻⁶ 6 Cells / kg, approximately 5 × 10⁻⁶ 6 Cells / kg, approximately 6 × 10⁻⁶ 6 Cells / kg, approximately 7 × 10 6 Cells / kg, approximately 8 × 10 6 Cells / kg, approximately 9 × 10⁻⁶ 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2 × 10⁻⁶ 7 Cells / kg, approximately 3 × 10⁻⁶ 7 cells / kg, approximately 4 × 10⁻⁶ 7 Cells / kg, approximately 5 × 10⁻⁶ 7 Cells / kg, approximately 6 × 10⁻⁶ 7 Cells / kg, approximately 7 × 10 7 Cells / kg, approximately 8 × 10 7 Cells / kg or approximately 9 × 10⁻⁶ 7 Cells / kg

[0193] In some implementation schemes, the therapeutically effective amount of engineered live T cells is between approximately 1 × 10⁻⁶ per kg of body weight. 6 With approximately 2×10 6 Between engineered live T cells up to approximately 1 × 10 8 The maximum dose of engineered live T cells.

[0194] In some implementations, the engineered T cells are anti-CD19 CAR T cells. In some implementations, the anti-CD19 CAR T cells are Achillenafil products, YESCARTA. ™ Axi-cel, TECARTUS ™ -Breki Orensay / KTE-X19, KYMRIAH ™ (Texavenylene), lecithin lecithin. In some implementations, the engineered T cells are anti-BCMACAR T cells, such as lecithin / bb2121, etc. In some implementations, the product meets commercial specifications. In some implementations, the product does not meet commercial specifications (non-compliant product, OOS). In some implementations, the OOS product contains fewer, less differentiated CCR7+ T cells compared to the commercially compliant lecithin product. N and T CM And a larger proportion of highly differentiated CCR7-T EM + T EFFCells. In some embodiments, the OOS product produces a median peak CAR T cell level that is lower than the median peak CAR T cell level of the commercial product following administration. In some embodiments, the OOS product still exhibits manageable safety profiles and meaningful clinical benefits.

[0195] The present application also provides a dose and administration of cells made by the methods of the present application, e.g., an infusion bag of a CD 19-directed genetically modified autologous T cell immunotherapy comprising a chimeric antigen receptor (CAR)-positive T cell suspension for infusion in about 68 mL. In some embodiments, the CAR T cells are formulated for infusion in about 40 mL. In some embodiments, the total volume of the CAR T cell product is formulated is 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 500 mL, 700 mL, 800 mL, 900 mL, 1000 mL. In one aspect, a dose and administration of cells made by the methods of the present application, e.g., an infusion bag of a CD 19-directed genetically modified autologous T cell immunotherapy comprises 1 x 10 6 CAR-T positive cells in about 40 mL. The target dose can be between about 1 x 10 6 and about 2 x 10 6 CAR positive viable T cells, with a maximum of 2 x 10 8 CAR positive viable T cells.

[0196] In some embodiments, the dosage form comprises a cell suspension for infusion in a single-use patient-specific infusion bag; the route of administration is intravenous; the entire contents of each single-use patient-specific bag is infused over 30 minutes by gravity or peristaltic pump. In one embodiment, the dosing regimen is a single infusion comprising 2.0 x 10 6 anti-CD 19 CAR T cells / kg body weight (± 20%), with a maximum dose of 2 x 10 8 anti-CD 19 CAR T cells (for subjects > 100 kg). In some embodiments, the T cells that make up the dose are CD 19 CAR-T cells.

[0197] Conditioning agents

[0198] In some embodiments, a conditioning agent is administered to the subject prior to the immunotherapy. In some embodiments, the conditioning is performed with radiation therapy. In some embodiments, the conditioning therapy is lymphodepleting chemotherapy.

[0199] In one embodiment, the conditioning therapy comprises an alkylating agent selected from the group consisting of melphalan, chlorambucil, cyclophosphamide, mechlorethamine, chlormethine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, an alkyl sulfonate, busulfan, thiotepa, or analogs thereof, and any combination thereof; a purine analog selected from the group consisting of azathioprine, 6-mercaptopurine, mercaptopurine, thiopurine, thioguanine, fludarabine, pentostatin, cladribine, and any combination thereof; and / or a platinum-based pre-conditioning agent selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazenes, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.

[0200] In another embodiment, the one or more pre-conditioning agents can comprise a platinum-based chemotherapy agent. In certain embodiments, the platinum-based chemotherapy agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazenes, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, any analog or functional derivative thereof, and any combination thereof.

[0201] In another embodiment, the one or more pre-conditioning agents can comprise a purine analog. In certain embodiments, the purine analog is selected from the group consisting of azathioprine, 6-mercaptopurine, mercaptopurine, thiopurine, thioguanine, fludarabine, pentostatin, cladribine, any analog or functional derivative thereof, and any combination thereof. In one embodiment, the one or more pre-conditioning agents comprise fludarabine.

[0202] In some embodiments, the one or more pre-conditioning agents can comprise cyclophosphamide and a purine analog. The purine analog can be selected from the group consisting of azathioprine, 6-mercaptopurine, mercaptopurine, thiopurine, thioguanine, fludarabine, pentostatin, cladribine, any analog or functional derivative thereof, and any combination thereof. In a particular embodiment, the one or more pre-conditioning agents comprise cyclophosphamide and pentostatin. In a particular embodiment, the one or more pre-conditioning agents comprise cyclophosphamide and fludarabine. The dosing amounts and dosing regimens for cyclophosphamide and fludarabine are described, by way of non-limiting example, in International Publication No. WO 2019 / 079564, International Publication No. WO 2021 / 092290, International Publication No. WO 2015 / 20096, and International Publication No. WO 2016 / 191755, each of which is incorporated herein by reference in its entirety.

[0203] In certain embodiments, a first dose (also applicable to repeat doses) of one or more preconditioning agents is administered to the patient. For example, in some embodiments, the first dose of cyclophosphamide is about 300 mg / m 2 / day to about 2000 mg / m 2 / day. In another embodiment, the first dose of cyclophosphamide is higher than 300 mg / m 2 / day and lower than 2000 mg / m 2 / day. In other embodiments, the dose of cyclophosphamide is about 350 mg / m 2 / day to about 2000 mg / m 2 / day, at least about 400 mg / m 2 / day to about 2000 mg / m 2 / day, about 450 mg / m 2 / day to about 2000 mg / m 2 / day, about 500 mg / m 2 / day to about 2000 mg / m 2 / day, about 550 mg / m 2 / day to about 2000 mg / m 2 / day, or about 600 mg / m 2 / day to about 2000 mg / m 2 / day. In other embodiments, the dose of cyclophosphamide is about 350 mg / m 2 / day to about 1500 mg / m 2 / day, about 350 mg / m 2 / day to about 1000 mg / m 2 / day, about 400 mg / m 2 / day to about 900 mg / m 2 / day, about 450 mg / m 2 / day to about 800 mg / m 2 / day, about 450 mg / m 2 / day to about 700 mg / m 2 / day, about 500 mg / m 2 / day to about 600 mg / m 2 / day, or about 300 mg / m 2 / day to about 500 mg / m 2 / day. In another embodiment, the dose of cyclophosphamide is about 350 mg / m 2 / day, about 400 mg / m 2 / day, about 450 mg / m 2 / day, about 500 mg / m 2 / day, about 550 mg / m 2 / day, about 600 mg / m2 / day, approximately 650mg / m² 2 / day, approximately 700mg / m 2 / day, approximately 800mg / m 2 / day, approximately 900mg / m 2 / day or approximately 1000mg / m 2 / sky.

[0204] In other embodiments, the first dose of cyclophosphamide (which also applies to repeated doses) is approximately 200 mg / m². 2 / day to approximately 3000mg / m 2 / day. In another embodiment, the first dose of cyclophosphamide is higher than 200 mg / m². 2 / day and below 3000mg / m 2 / day. In other embodiments, the dose of cyclophosphamide is approximately 200 mg / m². 2 / day - approximately 3000mg / m 2 / day, approximately 300mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 400mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 500mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 600mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 700mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 800mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 900mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1000mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1100mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1200mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1300mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1400mg / m 2 / day - approximately 3000mg / m 2 / day, approximately 1500mg / m 2about 3000 mg / m 2 about 1600 mg / m 2 about 3000 mg / m 2 about 1700 mg / m 2 about 3000 mg / m 2 about 1800 mg / m 2 about 3000 mg / m 2 about 1900 mg / m 2 about 3000 mg / m 2 about 2000 mg / m 2 about 3000 mg / m 2 about 200 mg / m 2 about 2900 mg / m 2 about 400 mg / m 2 about 2800 mg / m 2 about 500 mg / m 2 about 2700 mg / m 2 about 600 mg / m 2 about 2600 mg / m 2 about 700 mg / m 2 about 2500 mg / m 2 about 800 mg / m 2 about 2400 mg / m 2 about 900 mg / m 2 about 2350 mg / m 2 about 1000 mg / m 2 about 2300 mg / m 2 about 1100 mg / m 2 about 2250 mg / m 2 about 1110 mg / m 2 about 2220 mg / m 2 about 1000 mg / m 2 about 300 mg / m 2 about 500 mg / m 2 about 1000 mg / m

[0205] about 20 mg / m 2 about 900 mg / m 2mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2 mg / m2 / day. In some embodiments, the dose of fludarabine is higher than 30 mg / m2 / day and lower than 900 mg / m2 / day. 2mg / m2 / day. In some embodiments, the dose of fludarabine is about 20 mg / m2 / day. 2 mg / m2 / day, about 25 mg / m2 / day 2 mg / m2 / day, about 30 mg / m2 / day 2 mg / m2 / day, about 35 mg / m2 / day 2 mg / m2 / day, about 40 mg / m2 / day 2 mg / m2 / day, about 45 mg / m2 / day 2 mg / m2 / day, about 50 mg / m2 / day 2 mg / m2 / day, about 55 mg / m2 / day 2 mg / m2 / day, about 60 mg / m2 / day 2 mg / m2 / day, about 65 mg / m2 / day 2 mg / m2 / day, about 70 mg / m2 / day 2 mg / m2 / day, about 75 mg / m2 / day 2 mg / m2 / day, about 80 mg / m2 / day 2 mg / m2 / day, about 85 mg / m2 / day 2 mg / m2 / day, about 90 mg / m2 / day 2 mg / m2 / day, about 95 mg / m2 / day 2 mg / m2 / day, about 100 mg / m2 / day 2 mg / m2 / day, about 200 mg / m2 / day 2 mg / m2 / day, or about 300 mg / m2 / day 2 mg / m2 / day. In some embodiments, the dose of fludarabine is about 20 mg / m2 / day. 2 mg / m2 / day, about 25 mg / m2 / day 2 mg / m2 / day, about 30 mg / m2 / day 2 mg / m2 / day, about 35 mg / m2 / day 2 mg / m2 / day, about 40 mg / m2 / day 2 mg / m2 / day, about 45 mg / m2 / day 2 mg / m2 / day, about 50 mg / m2 / day 2 mg / m2 / day, about 55 mg / m2 / day 2 mg / m2 / day, about 60 mg / m2 / day 2 mg / m2 / day, about 65 mg / m2 / day 2 mg / m2 / day, about 70 mg / m2 / day 2 mg / m2 / day, about 75 mg / m2 / day 2 mg / m2 / day, about 80 mg / m2 / day 2 mg / m2 / day, about 85 mg / m2 / day 2 mg / m2 / day, about 90 mg / m2 / day 2 mg / m2 / day, about 95 mg / m2 / day 2 mg / m2 / day, or about 100 mg / m2 / day 2 mg / m2 / day. In other embodiments, the dose of fludarabine is about 110 mg / m2 / day. 2 mg / m2 / day, 120 mg / m2 / day 2 mg / m2 / day, 130 mg / m2 / day 2 mg / m2 / day, 140 mg / m2 / day 2 mg / m2 / day, 150 mg / m2 / day 2 / day, 160mg / m 2 / day, 170mg / m 2 / day, 180mg / m 2 / day or 190mg / m 2 / day. In some implementations, the dose of fludarabine is approximately 210 mg / m². 2 / day, 220mg / m 2 / day, 230mg / m 2 / day, 240mg / m 2 / day, 250mg / m 2 / day, 260mg / m 2 / day, 270mg / m 2 / day, 280mg / m 2 / day or 290mg / m 2 / day. In one particular implementation, the dose of fludarabine is approximately 20 mg / m². 2 / day. In one particular implementation, the dose of fludarabine is approximately 25 mg / m². 2 / day. In another implementation, the dose of fludarabine is approximately 30 mg / m². 2 / day. In another implementation, the dose of fludarabine is approximately 60 mg / m². 2 / sky.

[0206] The timing of administration of one or more preconditioning agents can be adjusted to maximize efficacy. In some embodiments, one or more preconditioning agents comprise two or more preconditioning agents. The two or more preconditioning agents can be administered simultaneously or sequentially. In a particular embodiment, (e.g., cyclophosphamide) is administered to the patient before or after a second preconditioning agent (e.g., fludarabine).

[0207] The doses of cyclophosphamide and fludarabine can be increased or decreased together or independently. For example, the dose of cyclophosphamide can be increased while the dose of fludarabine is decreased, and the dose of cyclophosphamide can be decreased while the dose of fludarabine is increased. Alternatively, the doses of both cyclophosphamide and fludarabine can be increased or decreased together. In some embodiments, the dose of cyclophosphamide is 300 mg / m². 2 / day, and the dose of fludarabine is 20mg / m². 2 / day. In other embodiments, the dose of cyclophosphamide is 300 mg / m². 2 / day, and the dose of fludarabine is 30mg / m². 2 / day. In other embodiments, the dose of cyclophosphamide is 300 mg / m². 2 / day, and the dose of fludarabine is 60mg / m². 2mg / m2 / day, and the dose of fludarabine is 20 mg / m2 / day. In other embodiments, the dose of cyclophosphamide is 500 mg / m2 / day, and the dose of fludarabine is 30 mg / m2 / day. In other embodiments, the dose of cyclophosphamide is 500 mg / m2 / day, and the dose of fludarabine is 60 mg / m2 / day. In other embodiments, the dose of cyclophosphamide is 200 mg / m2 / day, and the dose of fludarabine is 20 mg / m2 / day. In other embodiments, the dose of cyclophosphamide is 200 mg / m2 / day, and the dose of fludarabine is 30 mg / m2 / day. In other embodiments, the dose of cyclophosphamide is 200 mg / m2 / day, and the dose of fludarabine is 60 mg / m2 / day. 2 2 2 2 2 2 2 2 2 2 2 2

[0208] As described herein, the day on which the T cell therapy is administered is designated as day 0. The one or more preconditioning agents can be administered at any time prior to administration of the T cell therapy. In some embodiments, administration of the one or more preconditioning agents begins at least seven days, at least six days, at least five days, at least four days, at least three days, at least two days, or at least one day prior to administration of the T cell therapy. In other embodiments, administration of the one or more preconditioning agents begins at least eight days, at least nine days, at least ten days, at least eleven days, at least twelve days, at least thirteen days, or at least fourteen days prior to administration of the T cell therapy. In one embodiment, administration of the one or more preconditioning agents begins about seven days prior to administration of the T cell therapy. In another embodiment, administration of the one or more preconditioning agents begins about five days prior to administration of the T cell therapy.

[0209] In one embodiment, administration of the first preconditioning agent begins about seven days prior to administration of the T cell therapy, and administration of the second preconditioning agent begins about five days prior to administration of the T cell therapy. In a particular embodiment, the first preconditioning agent is administered to the patient for two days, about seven days and about six days prior to administration of the T cell therapy. In another embodiment, the second preconditioning agent is administered to the patient for five days, about five days, four days, three days, two days, and one day prior to administration of the T cell therapy. In another embodiment, the first preconditioning agent is administered to the patient for three days, about five days, four days, and three days prior to administration of the T cell therapy.

[0210] ​​​​​​​​​​​​In a particular embodiment, the administration of cyclophosphamide begins about seven days prior to the administration of the T cell therapy, and the administration of the purine analog (e.g., fludarabine or pentostatin) begins about five days prior to the administration of the T cell therapy. In another embodiment, the administration of cyclophosphamide begins about five days prior to the administration of the T cell therapy, and the administration of the purine analog (e.g., fludarabine or pentostatin) begins about five days prior to the administration of the T cell therapy.

[0211] The timing of administration of each component can be adjusted to maximize the effect. Generally, one or more pre-conditioning agents can be administered daily. In some embodiments, one or more pre-conditioning agents are administered daily for about two days, about three days, about four days, about five days, about six days, or about seven days. In some embodiments, one or more pre-conditioning agents can be administered daily for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. In a particular embodiment, one or more pre-conditioning agents are administered daily for about three days.

[0212] As described herein, the day that the T cell therapy is administered to the patient is designated as day 0. In some embodiments, one or more pre-conditioning agents, e.g., cyclophosphamide, are administered to the patient on day -7 and day -6 (i.e., days -7 and -6) prior to day 0. In other embodiments, one or more pre-conditioning agents, e.g., cyclophosphamide, are administered to the patient on day -5, day -4, and day -3. In some embodiments, one or more pre-conditioning agents, e.g., fludarabine, are administered to the patient on day -5, day -4, day -3, day -2, and day -1. In other embodiments, one or more pre-conditioning agents, e.g., fludarabine, are administered to the patient on day -5, day -4, and day -3.

[0213] One or more pre-conditioning agents, e.g., cyclophosphamide and fludarabine, can be administered on the same or different days. If cyclophosphamide and fludarabine are administered on the same day, the cyclophosphamide dose can be administered before or after the fludarabine dose. In one embodiment, a cyclophosphamide dose is administered to the patient on day -7 and day -6, and a fludarabine dose is administered to the patient on day -5, day -4, day -3, day -2, and day -1. In another embodiment, a cyclophosphamide dose is administered to the patient on day -5, day -4, and day -3, and a fludarabine dose is administered to the patient on day -5, day -4, and day -3.

[0214] In certain embodiments, one or more pre-conditioning agents, e.g., cyclophosphamide and fludarabine, can be administered simultaneously or sequentially. In one embodiment, cyclophosphamide is administered to the patient before fludarabine. In another embodiment, cyclophosphamide is administered to the patient after fludarabine.

[0215] The route of administration and the administration regimen of the one or more pre-conditioning agents are known in the art, for example described at least in International Publication No. WO 2019 / 079564, International Publication No. WO 2021 / 092290, International Publication No. WO 2015 / 20096, and International Publication No. WO 2016 / 191755, each of which is incorporated by reference herein in its entirety.

[0216] Cancer

[0217] The methods disclosed herein can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor in a patient, prevent recurrence of a tumor, prevent metastasis of a tumor, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0218] Cancers that can be treated include tumors that are not vascularized, not yet sufficiently vascularized, or vascularized. The cancer can also include a solid tumor or a non-solid tumor. In some embodiments, the cancer is a cancer of white blood cells. In other embodiments, the cancer is a cancer of plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute lymphatic leukemia (ALL) and hemophagocytic lymphohistiocytosis (HLH), B-cell prolymphocytic leukemia, B-cell acute lymphatic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic or acute granulomatous disease, chronic or acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, hemophagocytic syndrome (macrophage activation syndrome (MAS), Hodgkin's disease, large cell granuloma, leukocyte adhesion deficiency, lymphoproliferative disorder of malignant lymphohistiocytic, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of unknown significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome (MDS), myeloid disease (including but not limited to acute myeloid leukemia (AML)), non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell tumor, plasmacytoma (e.g., plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome; Takatsuki disease; PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell- or large cell-follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphatic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenstrom macroglobulinemia, DLBCL arising from FL, high-grade B-cell lymphoma, or a combination thereof.

[0219] In some implementations, the cancer is myeloma. In some implementations, the cancer is multiple myeloma. In some implementations, the cancer is leukemia. In some implementations, the cancer is acute myeloid leukemia. In some implementations, the cancer is relapsed or refractory large B-cell lymphoma (possibly after two or more lines of systemic therapy), including nonspecific diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, or relapsed or refractory follicular lymphoma (FL) (possibly after two or more lines of systemic therapy), or relapsed or refractory mantle cell lymphoma (MCL).

[0220] In some implementations, the cancer is non-Hodgkin's lymphoma. In some implementations, the cancer is relapsed / refractory NHL. In some implementations, the cancer is mantle cell lymphoma.

[0221] In some implementations, the cancer is advanced indolent non-Hodgkin lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL). In some implementations, the patient has relapsed / refractory disease after ≥2 prior lines of therapy, including anti-CD20 monoclonal antibodies with alkylating agents. In some implementations, the patient may have received a PI3K inhibitor. In some implementations, the patient may (also) have received an autologous stem cell transplant. In some implementations, the patient first undergoes leukoablation to obtain T cells for CAR T cell preparation, followed by conditioning chemotherapy with cyclophosphamide 500 mg / m². 2 / day, fludarabine 30mg / m 2 / day, administered on days -5, -4, and -3; on day 0, patients may receive a single intravenous infusion of CAR T-cell therapy (e.g., acilantroxetine, benotuximab) with a target dose of 2 × 10⁶. 6 CAR T cells / kg. In some embodiments, an additional infusion may be given at a later time. In some embodiments, if a patient progresses after a response at the assessment at 3 months following the initial administration, the patient may be retreated with CAR T cell therapy (e.g., azithromycin). In some embodiments, the patient may receive bridging therapy. Examples of bridging therapies are provided elsewhere in the specification (including examples). In some embodiments, the patient experiences CRS. In some embodiments, CRS is managed using any of the protocols described in this application (including examples). In some embodiments, CRS is managed with tocilizumab, corticosteroids, and / or vasopressors.

[0222] In some embodiments, the cancer is relapsed / refractory indolent non-Hodgkin lymphoma and the method of treating a subject in need thereof comprises administering to the subject a therapeutically effective amount of CAR T cells as a retreatment, wherein the subject has previously received a first treatment with CAR T cells. In some embodiments, the first treatment with CAR T cells can be administered as a first line therapy or a second line therapy, optionally wherein the lymphoma is R / R follicular lymphoma (FL) or marginal zone lymphoma (MZL), and optionally wherein the previous number of lines of therapy includes an anti-CD20 monoclonal antibody in combination with an alkylating agent. In some embodiments, the conditioning therapy comprises intravenous infusion of fludarabine 30 mg / m2 / day on days -5, -4, and -3, and cyclophosphamide 500 mg / m2 / day on days -5, -4, and -3. 2 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 2 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 4 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 5 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 8 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 9 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 10 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 4 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 5 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 8 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 6 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 7 In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 107 Cells / kg, approximately 7 × 10 7 Cells / kg, approximately 8 × 10 7 Cells / kg or approximately 9 × 10⁻⁶ 7 Cells / kg. In some embodiments, the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cells are acerencin CAR T cells. In some embodiments, the criteria for retreatment eligibility include a CR or PR response at month 3 disease assessment and subsequent progression; no evidence of CD19 loss in the progression biopsy by local examination; and / or no grade 4 CRS or neurological event, or life-threatening toxicity, at the time of initial CAR T cell treatment. In some embodiments, the treatment follows the protocol adopted in the clinical trial (NCT03105336).

[0223] In some implementations, the cancer is NHL and immunotherapy (e.g., CAR T or TCR T-cell therapy) is administered as first-line therapy. In some implementations, the cancer is LBCL. In some implementations, LBCL is high-risk / high-grade LBCL with MYC and BCL2 and / or BCL6 translocations or DLBCL with an IPI score ≥3 at any time prior to enrollment. In some implementations, first-line therapy includes a combination of CAR T-cell therapy with an anti-CD20 monoclonal antibody and an anthracycline-containing regimen. In some implementations, CAR T-cell therapy is administered first. In some implementations, an anti-CD20 monoclonal antibody / anthracycline-containing regimen is administered first. In some implementations, treatment is administered for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at intervals of less than one year, etc. In some implementations, the method also includes a bridging therapy administered after leukapheresis and completed before initiating conditioning chemotherapy. In some implementations, additional inclusion criteria include age ≥18 years and ECOG PS 0-1. In some implementations, conditioning therapy includes intravenous infusion of fludarabine 30 mg / m² on days -5, -4, and -3. 2 Combined intravenous infusion of cyclophosphamide 500 mg / m 2 Other exemplary and beneficial preconditioning treatment regimens are described in U.S. Provisional Patent Applications 62 / 262,143 and 62 / 167,750 and U.S. Patent Nos. 9,855,298 and 10,322,146, the entire contents of which are incorporated herein by reference. These provisional patent applications describe, for example, methods for conditioning patients requiring T-cell therapy, including administering to the patient a specified beneficial dose of cyclophosphamide (200 mg / m²). 2 / day and 2000mg / m 2between about 200 mg / m2 / day and about 300 mg / m2 / day of cyclophosphamide and between about 20 mg / m2 / day and about 50 mg / m2 / day of fludarabine for three days. In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 107CAR T cells / kg on day 0. In some embodiments, at least about 10 2 cells, at least about 10 2 cells, at least about 10 2 cells, at least about 10 2 cells, at least about 10 2 cells, at least about 10 2 cells, or at least about 10 2 cells are administered. In another embodiment, the therapeutically effective amount of T cells is about 10 2 cells, about 10 2 cells, about 10 2 cells, about 10 2 cells, or about 10 2 cells. Other conditioning regimens include 200 mg / m2 / day to 300 mg / m2 / day of cyclophosphamide and 20 mg / m2 / day to 50 mg / m2 / day of fludarabine for three days. In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 107CAR T cells / kg on day 0. In some embodiments, at least about 10 2 cells, at least about 10 2 cells, at least about 10 6 cells, at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, or at least about 10 8 cells are administered. In another embodiment, the therapeutically effective amount of T cells is about 10 9 cells, about 10 10 cells, about 10 4 cells, about 10 5 cells, about 10 6 cells, or about 10 7 cells.8 1010 cells. In some implementations, the therapeutically effective amount of T cells is approximately 2 × 1010. 6 Cells / kg, approximately 3 × 10⁻⁶ 6 cells / kg, approximately 4 × 10⁻⁶ 6 Cells / kg, approximately 5 × 10⁻⁶ 6 Cells / kg, approximately 6 × 10⁻⁶ 6 Cells / kg, approximately 7 × 10 6 Cells / kg, approximately 8 × 10 6 Cells / kg, approximately 9 × 10⁻⁶ 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2 × 10⁻⁶ 7 Cells / kg, approximately 3 × 10⁻⁶ 7 cells / kg, approximately 4 × 10⁻⁶ 7 Cells / kg, approximately 5 × 10⁻⁶ 7 Cells / kg, approximately 6 × 10⁻⁶ 7 Cells / kg, approximately 7 × 10 7 Cells / kg, approximately 8 × 10 7 Cells / kg or approximately 9 × 10⁻⁶ 7 Cells / kg. In some embodiments, CAR T cells are anti-CD19 CAR T cells. In some embodiments, CAR T cell therapy includes anti-CD19 CAR T cells. In some embodiments, CAR T cell therapy includes acetylene or yescarta. ™ In some implementations, CAR T-cell therapy includes TECARTUS. ™ —Benotoxin or KYMRIAH ™ (Tisharunsai) etc., Akiwirunsai / bb2121.

[0224] In another embodiment, this disclosure provides a method of treating cancer in a subject of need, the method comprising administering a therapeutically effective amount of CD19 CAR-T therapy to a subject having received 1-2, 3, 4, or ≥5 lines of prior therapy. In one embodiment, this disclosure provides a method of treating cancer in a subject of need, the method comprising administering a therapeutically effective amount of CD19 CAR-T therapy to a subject having received 1-2 lines of prior therapy. The cancer may be any of the cancers listed above. The CD19 CAR-T therapy may be any of the CD19 CAR-T therapies listed above. In some embodiments, CD19 CAR-T therapy is used as first-line treatment. In some embodiments, CD19 CAR-T therapy is used as second-line treatment.

[0225] In an embodiment, the CD19 CAR-T therapy is any of the above CD19 CAR-T therapies. In an embodiment, the CD19 CAR-T therapy comprises axicabtagene ciloleucel treatment. In embodiments, the cancer is refractory DLBCL (ABC / GCB) not otherwise specified, HGBL with or without MYC and BCL2 and / or BCL6 rearrangement, DLBCL arising from FL, T-cell / histiocyte-rich large B-cell lymphoma, DLBCL associated with chronic inflammation, primary cutaneous DLBCL, leg type, and / or Epstein-Barr virus (EBV) + DLBCL. In an embodiment, the subject selected for axicabtagene ciloleucel treatment has refractory DLBCL (ABC / GCB) not otherwise specified, HGBL with or without MYC and BCL2 and / or BCL6 rearrangement, DLBCL arising from FL, T-cell / histiocyte-rich large B-cell lymphoma, DLBCL associated with chronic inflammation, primary cutaneous DLBCL, leg type, and / or Epstein-Barr virus (EBV) + DLBCL. In some embodiments, axicabtagene ciloleucel treatment is used as a second line therapy, where the first line therapy is CHOP, i.e., cyclophosphamide (Cytoxan ® ), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin ® ), and prednisone. In some embodiments, axicabtagene ciloleucel treatment is used as a second line therapy, where the first line therapy is R-CHOP (CHOP plus rituximab).

[0226] In embodiments, patients with relapsed or refractory disease after first line chemoimmunotherapy are selected for second line axicabtagene ciloleucel treatment, refractory disease is defined as no complete remission to first line therapy; individuals who are intolerant to first line therapy are excluded. Progressive disease (PD) is best response to first line therapy, stable disease (SD) is best response after at least 4 cycles of first line therapy (e.g., 4 cycles of R-CHOP), partial response (PR) is best response after at least 6 cycles, and biopsy-proven residual disease or disease progression < 12 months of therapy, and / or relapsed disease is defined as complete remission to first line therapy followed by biopsy-proven relapse < 12 months of first line therapy. In some embodiments, conditioning therapy is provided to patients selected for second line axicabtagene ciloleucel treatment, comprising intravenous infusion of fludarabine 30 mg / m 2 in combination with intravenous cyclophosphamide 500 mg / m 2 In some embodiments, axicabtagene ciloleucel treatment is used as a second line therapy.

[0227] Combination therapy

[0228] The compositions comprising immune effector cells expressing a CAR disclosed herein can be administered in combination with any number of chemotherapeutic agents (prior to, after, and / or concurrently with the T cell administration). In some embodiments, the antigen binding molecule, the transduced (or otherwise engineered) cell (such as a CAR), and the chemotherapeutic agent are each administered in an amount effective to treat the disease or disorder in the subject.Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thiopurine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamide; mitoguazone; mitomycin C; mycobacterial cell wall extract; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex; razoxane; sizofiran; spiroglycol; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; "Ara-C" (Cytosine arabinoside); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®. ™ , Bristol-Myers Squibb) and docetaxel (TAXOTERE® , Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as Targretin ™ (bexarotene), Panretin ™ (alevitinic acid); ONTAK ™ (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in combination with an anti-hormonal agent for the regulation or inhibition of hormone effects on tumors, such as anti-estrogens including, for example, tamoxifen, raloxifene, 4(5)-imidazoles that inhibit aromatase, 4-hydroxytamoxifen, trioxifene, droloxifene, LY117018, onapristone, and toremifene (fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including but not limited to CHOP, i.e., cyclophosphamide (Cytoxan ® ), doxorubicin (adriamycin), vincristine (Oncovin ® ), and prednisone, R-CHOP (CHOP plus rituximab) and G-CHOP (CHOP plus obinutuzumab).

[0229] In some embodiments, the chemotherapeutic agent is administered at the same time as or within one week of administration of the engineered cells. In other embodiments, the chemotherapeutic 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 chemotherapeutic agent is administered at least 1 month prior to administration of the cells or nucleic acids. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0230] A variety of additional therapeutic agents can be used in conjunction with the compositions described herein (prior to, after and / or simultaneously with T cell administration). For example, potential useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO ® ), pembrolizumab (KEYTRUDA® ), Cemiplimab (Libtayo), pidilizumab (CureTech), and atezolizumab (Roche), and PD-L1 inhibitors such as atezolizumab, durvalumab, and avelumab. In some embodiments, the therapeutic agent used in combination is an anti-IL-1 (e.g., anakinra), a T cell activation inhibitor (e.g., dasatinib), a JAK inhibitor (e.g., filgotinib), an anti-GM-CSF (e.g., lenzilumab), an anti-TNF (e.g., infliximab), an Ang2 inhibitor (e.g., azilsartan), an anti-angiogenic therapy (e.g., bevacizumab), and / or an anti-IFNg (e.g., ipilimumab).

[0231] Additional therapeutic agents suitable for use in combination (prior to, after, and / or concurrently with the T cell administration) with the compositions and methods disclosed herein include, but are not limited to, Ibrutinib (IMBRUVICA ® ), Ofatumumab (ARZERRA ® ), Rituximab (RITUXAN ® ), Bevacizumab (AVASTIN ® ), Trastuzumab (HERCEPTIN ® ), Enfortumab vedotin (KADCYLA ® ), Imatinib (GLEEVEC ® ), Cetuximab (ERBITUX ® ), Panitumumab (VECTIBIX ® ), Catumaxomab, Ibritumomab tiuxetan, Ofatumumab, Tositumomab, Bendamustine, Alemtuzumab, Gemtuzumab, Erlotinib, Gefitinib, Vandetanib, Afatinib, Lapatinib, Nilotinib, Masitinib, Pazopanib, Sunitinib, Sorafenib, Tivantinib, Axitinib, Cediranib, Ramucirumab, Regorafenib, Semaxanib, Sorafenib, Sunitinib, Tivozanib, Tivantinib, Vandetanib, Encorafenib, Cabozantinib, Imatinib, Dasatinib, Nilotinib, Ponatinib, Radotinib, Bosutinib, Tivantinib, Ruxolitinib, Pacritinib, Cobimetinib, Semapimib, Trametinib, Binimetinib, Alectinib, Cetuximab, Crizotinib, Aflibercept, Adipotide, Denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib), inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, and anti-thymocyte globulin, lenzilumab, and mavrilimumab.

[0232] In one embodiment, the GM-CSF inhibitor is selected from lenzilumab; narsoplimab (AMG203); GSK3196165 / MOR103 / ocetixumab (GSK / MorphoSys); KB002 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022 / rimonab (Morphotek); or a biosimilar of any of them; E21R; and a small molecule. In one embodiment, the CSF1 inhibitor is selected from RG7155, PD-0360324, MCS110 / latrunculin A or a biosimilar version of any of them; and a small molecule. In one embodiment, the GM-CSFR inhibitor and the CSF1R inhibitor are selected from mavrilimumab (formerly CAM-3001; Medlmmune, Inc.); cabiralizumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly and Company), emactuzumab, also known as RG7155 or RO5509554; FPA008 (Five Prime / BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041 / AFS98 / TG3003 (ElsaLys Bio, Transgene), SNDX-6352 (Syndax); a biosimilar version of any of them; and a small molecule.

[0233] In some embodiments, the compositions comprising immunotherapy (e.g., engineered CAR T cells) are administered with an anti-inflammatory agent (prior to, after, and / or concurrently with T cell administration). Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), infliximab (REMICADE®), certolizumab (CIMZIA®), and golimumab (SIMPONI®)), and interleukin-1 receptor antagonists (e.g., rilonacept (ARCALIA®) and canakinumab (ILARIS®)).® adalimumab (HUMIRA ® ) and infliximab (REMICADE ® ), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold salts (oral (auranofin) and intramuscular), and minocycline.

[0234] In some embodiments, the compositions described herein are administered in combination with a cytokine (prior to, after, or simultaneously with T cell administration). Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-β; platelet- growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO, Epogen ® , Procrit ® ); osteoinductive factors; interferons such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15; tumor necrosis factor-alpha and -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.

[0235] In some embodiments, the administration of the cells and the administration of the additional therapeutic agent are performed on the same day, within no more than 36 hours, within no more than 24 hours, within no more than 12 hours, within no more than 6 hours, within no more than 4 hours, within no more than 2 hours, or within no more than 1 hour or within no more than 30 minutes. In some embodiments, the administration of the cells and the administration of the additional therapeutic agent are performed at a time that is between or about 0 hours and between or about 48 hours, between or about 0 hours and between or about 36 hours, between or about 0 hours and between or about 24 hours, between or about 0 hours and between or about 12 hours, between or about 0 hours and between or about 6 hours, between or about 0 hours and between or about 2 hours, between or about 0 hours and between or about 1 hour, between or about 0 minutes and between or about 30 minutes, between or about 30 minutes and between or about 48 hours, between or about 30 minutes and between or about 36 hours, between or about 30 minutes and between or about 24 hours, between or about 30 minutes and between or about 12 hours, between or about 30 minutes and between or about 6 hours, between or about 30 minutes and between or about 4 hours, between or about 30 minutes and between or about 2 hours, between or about 30 minutes and between or about 1 hour, between or about 1 hour and between or about 48 hours, between or about 1 hour and between or about 36 hours, between or about 1 hour and between or about 24 hours, between or about 1 hour and between or about 12 hours, between or about 1 hour and between or about 6 hours, between or about 1 hour and between or about 4 hours, between or about 1 hour and between or about 2 hours, between or about 2 hours and between or about 48 hours, between or about 2 hours and between or about 36 hours, between or about 2 hours and between or about 24 hours, between or about 2 hours and between or about 12 hours, between or about 2 hours and between or about 6 hours, between or about 2 hours and between or about 4 hours, between or about 4 hours and between or about 48 hours, between or about 4 hours and between or about 36 hours, between or about 4 hours and between or about 24 hours, between or about 4 hours and between or about 12 hours, between or about 4 hours and between or about 6 hours, between or about 6 hours and between or about 48 hours, between or about 6 hours and between or about 36 hours, between or about 6 hours and between or about 24 hours, between or about 6 hours and between or about 12 hours, between or about 12 hours and between or about 48 hours, between or about 12 hours and between or about 36 hours, between or about 12 hours and between or about 24 hours, between or about 24 hours and between or about 48 hours, between or about 24 hours and between or about 36 hours, or between or about 36 hours and between or about 48 hours. In some embodiments, the cells and the additional therapeutic agent are administered simultaneously.

[0236] In some embodiments, the agent is administered at a dose equal to or about 30 mg to 5000 mg, such as 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 500 mg, 100 mg to 200 mg, 200 mg to 1000 mg, 200 mg to 500 mg, or 500 mg to 1000 mg.

[0237] In some embodiments, the agent is administered at a dose of 0.5 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 25 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 5 mg / kg to 100 mg / kg, 5 mg / kg to 50 mg / kg, 5 mg / kg to 25 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, 10 mg / kg to 25 mg / kg, 25 mg / kg to 100 mg / kg, 25 mg / kg to 50 mg / kg to 50 mg / kg to 100 mg / kg. In some embodiments, the agent is administered at a dose of each 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg, 2 mg / kg to 4 mg / kg, or 6 mg / kg to 8 mg / kg. In some aspects, the agent is administered at a dose of at least 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, or more.

[0238] In some embodiments, the agent is administered by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, sub-tenon injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonarily, and intranasally, and if local therapy is desired, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0239] In some embodiments, the treatment further comprises a bridging therapy, which is a therapy modulating between the compositions disclosed herein or a therapy administered after leukapheresis and completed prior to initiation of conditioning chemotherapy. In some embodiments, the bridging therapy comprises CHOP, G-CHOP, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), corticosteroids, bendamustine, platinum compounds, anthracyclines, and / or phosphoinositide 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pictilisib (GDC 0941), copanlisib, PX 866, buparlisib (BKM120), pilaralisib (XL 147), GNE 317, Alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and Taselisib (GDC 0032). In some embodiments, the AKT inhibitor is perifosine, MK-2206. In one embodiment, the mTOR inhibitor is selected from everolimus, sirolimus, temsirolimus, ridafumos. In some embodiments, the dual PI3K / mTOR inhibitor is selected from BEZ235, XL765, and GDC-0980. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pictilisib (GDC 0941), copanlisib, PX 866, buparlisib (BKM120), pilaralisib (XL 147), GNE 317, Alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and Taselisib (GDC 0032).

[0240] In some embodiments, the bridge therapy comprises acalabrutinib, brentuximab vedotin, copanlisib hydrochloride, nirlaparib, belinostat, bendamustine hydrochloride, carmustine, bleomycin sulfate, bortezomib, zanabrutinib, carmustine, chlorambucil, copanlisib hydrochloride, denileukin diftitox, dexamethasone, doxorubicin hydrochloride, duvelisib, pralatrexate, obinutuzumab, ibritumomab tiuxetan, ibrutinib, idelalisib, recombinant interferon alfa-2b, romidepsin, lenalidomide, mechlorethamine hydrochloride, methotrexate, mogamulizumab-kpc, plerixafor, nirlaparib, obinutuzumab, denileukin diftitox, pembrolizumab, plerixafor, polatuzumab vedotin-piiq, mogamulizumab-kpc, prednisone, rituximab, hyaluronidase, romidepsin, bortezomib, venetoclax, vinblastine sulfate, vorinostat, zanabrutinib, CHOP, COPP, CVP, EPOCH, R-EPOCH, HYPER-CVAD, ICE, R-ICE, R-CHOP, R-CVP, and combinations thereof.

[0241] In some embodiments, the cellular immunotherapy is administered in combination with a debulking therapy for the purpose of reducing tumor burden. In one embodiment, the debulking therapy is administered after leukapheresis and prior to administration of conditioning chemotherapy or cell infusion. Examples of debulking therapies include the following (Table 1)

[0242] Table 1: Exemplary reduced-intensity bridge therapy

[0243]

[0244] Abbreviations: AUC, area under the curve

[0245] a Other debulking treatment options can be used and discussed with the medical monitor. Supportive treatments such as hydration, antiemetics, mesna, growth factor support, and tumor lysis prophylaxis can be used according to local standards. More than 1 cycle is allowed.

[0246] b At least 1 target lesion should be left outside the radiation field to allow tumor measurements

[0247] Monitoring

[0248] In some embodiments, administration of the immunotherapy (e.g., chimeric receptor T cell immunotherapy) is performed at an accredited medical facility.

[0249] In some embodiments, the methods disclosed herein include monitoring the patient for signs and symptoms of CRS and neurotoxicity and other adverse effects of the CAR T cell treatment at least daily for 7 days at an accredited medical facility following infusion. In some embodiments, the symptoms of neurotoxicity are selected from the group consisting of encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, the symptoms of adverse effects are selected from the group consisting of 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), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, the patient is instructed to remain near the accredited medical facility for at least 4 weeks following infusion.

[0250] Prevention or management of adverse events

[0251] In some embodiments, the methods include managing adverse events in any subject. The terms "adverse event," "adverse reaction," and "adverse effect" are used interchangeably herein. In some embodiments, the adverse event is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, severe infections, cytopenias, and hypogammaglobulinemia.

[0252] In some embodiments, the disclosure provides methods of preventing the occurrence of or mitigating the severity of adverse events based on the levels of a plurality of biomarkers in the serum of a subject receiving immunotherapy. In some embodiments, the cell therapy is administered with one or more agents that prevent, delay the onset of, reduce the symptoms of, or treat adverse events, whereby the adverse events include cytokine release syndrome and neurotoxicity. In one embodiment, the agents have been described above. In other embodiments, the agents are described below. In some embodiments, the agents are administered prior to, after, or concurrently with the administration of the cell by one of the methods and dosages described elsewhere in this specification. In one embodiment, the agent is administered to a subject who can be susceptible to the disease but has not yet been diagnosed with the disease.

[0253] In some embodiments, the immunotherapy (e.g., cell therapy) is administered prior to, during / simultaneously with, and / or after administration of one or more agents (e.g., steroids) or treatments (e.g., plaque excision) that treat and / or prevent (prophylactically) one or more symptoms of an adverse event. The pharmacological and / or physiological effect can be prophylactic, i.e., the effect completely or partially prevents the disease or its symptoms. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. In one embodiment, a prophylactically effective amount is used in a subject prior to or at the early stages of the disease. In one embodiment, a prophylactically effective amount will be less than a therapeutically effective amount. In some embodiments, patients are selected for management of adverse events based on expression of one or more markers described herein in the specification. In one embodiment, any patient who will receive, is symptomatic for, or has received cell therapy is administered an adverse event treatment or prevention.

[0254] In some embodiments, the signs and symptoms of adverse reactions are selected from 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), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0255] In some embodiments, patients are identified and selected based on one or more of the biomarkers described in the present application. In some embodiments, patients have been identified and selected simply by clinical presentation (e.g., presence and level of toxicity symptoms).

[0256] In some embodiments, the adverse event / reaction can be selected from one or more of the following (Table 2):

[0257] Table 2: Exemplary adverse events

[0258]

[0259]

[0260] Cytokine release syndrome (CRS)

[0261] In some embodiments, the method comprises 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.

[0262] In some embodiments, the method comprises identifying CRS based on clinical presentation. In some embodiments, the method comprises evaluating and treating other causes of fever, hypoxia, and hypotension. Patients who develop > grade 2 CRS (e.g., hypotension, hypoxia unresponsive to fluid resuscitation, or requiring supplemental oxygen) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients who develop severe CRS, consider performing echocardiogram to assess cardiac function. For severe or life-threatening CRS, intensive supportive care therapy can be considered.

[0263] In some embodiments, the method comprises monitoring the patient for signs and symptoms of CRS at least daily for 7 days at an accredited medical facility following infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of CRS for 4 weeks following infusion. In some embodiments, the method comprises advising the patient to seek immediate medical attention if signs or symptoms of CRS arise at any time. In some embodiments, treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids is administered upon the first sign of CRS.

[0264] In some embodiments, the subject experiences grade 3+ CRS. In some embodiments, this includes fever, hypotension, tachycardia, hypoxia, chills, sinus tachycardia, fatigue, headache, vomiting, acute kidney injury, myalgia, atrial fibrillation, diarrhea, dyspnea, decreased ejection fraction, pulmonary edema, atrial flutter, increased creatine, capillary leak syndrome, loss of appetite, febrile neutropenia, malaise, metabolic acidosis, fever, nausea, headache, rash, rapid heartbeat, hypotension, labored breathing, etc.

[0265] Neurotoxicity (NT)

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

[0267] In some embodiments, the subject experiences grade 3+ NT. In some embodiments, this includes encephalopathy, tremor, confusional state, aphasia, somnolence, agitation, memory impairment, dysarthria, hallucinations, altered mental status, ataxia, agitation, seizures, delirium, attention disturbances, somnolence, decreased level of consciousness, disorientation, calculation disturbances, hemiparesis, myoclonus, brain edema, etc.

[0268] Management of adverse events

[0269] In some embodiments, the method of managing adverse events comprises monitoring the patient for signs and symptoms of neurotoxicity at least daily for 7 days at an accredited medical facility following infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity and / or CRS for 4 weeks following infusion.

[0270] In some embodiments, the disclosure provides two methods of managing adverse events in subjects receiving steroid and anti-IL6 / anti-IL-6R antibody with CAR T cell therapy. In one embodiment, the CAR T cell therapy is anti-CD 19 therapy as described in the Examples. In one embodiment, the CAR T cell therapy is referred to as ZUMA-1, which includes different adverse event management protocols for different cohorts. In one embodiment, the disclosure demonstrates that early steroid intervention in cohort 4 is associated with a lower incidence of severe CRS and neurologic events compared to that observed in cohorts 1+2. In one embodiment, the disclosure demonstrates that early use of steroids in cohort 4 is associated with about 15% reduction in median cumulative corticosteroid equivalent dose in cohort 1+2, suggesting that early steroid use can allow for a reduction in overall steroid exposure. Accordingly, in one embodiment, the disclosure provides a method of managing adverse events, wherein corticosteroid therapy is initiated for all cases of grade 1 CRS if there is no improvement after 3 days and for all > grade 1 neurologic events. In one embodiment, tocilizumab is initiated for all cases of grade 1 CRS if there is no improvement after 3 days and for all > grade 2 neurologic events. In one embodiment, the disclosure provides a method of reducing overall steroid exposure in patients receiving adverse event management following CAR T cell administration, the method comprising initiating corticosteroid therapy for all cases of grade 1 CRS if there is no improvement after 3 days and for all > grade 1 neurologic events, and / or initiating tocilizumab for all cases of grade 1 CRS if there is no improvement after 3 days and for all > grade 2 neurologic events. In one embodiment, the corticosteroid and tocilizumab are administered in a regimen selected from those exemplified in Regimens A-C. In one embodiment, the disclosure demonstrates that earlier steroid use is not associated with an increased risk of severe infections, reduced CAR T cell expansion, or reduced tumor response.

[0271] In one embodiment, the present disclosure supports levetiracetam prophylaxis to prevent safety 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 axicabtagene ciloleucel. Accordingly, in one embodiment, the present disclosure provides a method of managing adverse events in a patient treated with CAR T cells, the method comprising administering to the patient a prophylactic dose of an anti-epileptic drug. In some embodiments, if a neurologic event occurs after discontinuation of prophylactic levetiracetam, the patient receives levetiracetam (e.g., 750 mg twice daily orally or intravenously) starting on day 0 of CAR T cell treatment (post-conditioning) and also upon 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, the levetiracetam prophylaxis is combined with any other adverse event management regimen.

[0272] In one embodiment, the present disclosure demonstrates that CAR T cell levels in patients receiving the adverse management regimen of Cohort 4 are comparable to those of Cohorts 1+2. In one embodiment, the present disclosure demonstrates that numerical levels of key inflammatory cytokines (e.g., IFNy, IL-2, and GM-CSF) associated with CAR-related inflammatory events are lower in Cohort 4 than in Cohorts 1+2. Accordingly, the present disclosure provides a method of reducing inflammatory events associated with CAR T cell treatment without impacting CAR T cell levels, the method comprising administering to the patient an adverse event management regimen of Cohort 4. The present disclosure also provides a method of reducing immune cell production of cytokines following CAR T cell therapy, the method comprising administering to the patient an adverse event management regimen of Cohort 4. In one embodiment, this effect is achieved without impacting CAR T cell expansion and response rate. In one embodiment, the patient has R / R LBCL. In one embodiment, the CAR T cell treatment is anti-CD19 CAR T cell treatment. In one embodiment, the CAR T cell treatment comprises axicabtagene ciloleucel.

[0273] In one embodiment, the present disclosure demonstrates that early or prophylactic use of tocilizumab for adverse event management after axicabtagene ciloleucel reduces > grade 3 cytokine release syndrome but increases > grade 3 neurologic events. Accordingly, the present disclosure provides a method for adverse event management in CAR T cell therapy. In one embodiment, the patient receives levetiracetam (750 mg twice daily orally or intravenously given) starting on day 0. At the onset of > grade 2 neurologic events, the levetiracetam dose is increased to 1000 mg twice daily. In one embodiment, if the patient does not experience any > grade 2 neurologic events, the levetiracetam is tapered and discontinued as clinically indicated. The patient also receives tocilizumab (8 mg / kg IV over 1 hour [not to exceed 800 mg]) on day 2. Further tocilizumab (± corticosteroids) can be recommended at the onset of grade 2 CRS in patients with comorbidities or advanced age or in the case of > grade 3 CRS. For patients who experience > grade 2 neurologic events, tocilizumab is initiated, and corticosteroids are added for patients with comorbidities or advanced age or if there is any occurrence of > grade 3 neurologic events despite the use of tocilizumab.

[0274] In one embodiment, the present disclosure demonstrates that prophylactic steroid use appears to reduce the rate of severe CRS and NE to a similar degree as steroid use in the early post-infusion period after axicabtagene ciloleucel administration. Accordingly, the present disclosure provides a method for managing adverse events in CAR T cell therapy, wherein the patient receives PO given dexamethasone 10 mg on day 0 (prior to axicabtagene ciloleucel infusion), day 1, and day 2. Steroids are also administered starting at grade 1 NE and grade 1 CRS when no improvement is observed after 3 days of supportive care. If no improvement is observed after 24 hours of supportive care, then tocilizumab is also administered for management of > grade 1 CRS.

[0275] In one embodiment, the present disclosure demonstrates that adverse event management of CAR T cell therapy with an antibody that neutralizes and / or depletes GM-CSF prevents or reduces treatment-related CRS and / or NE in the treated patient. In one embodiment, the antibody is otilimab.

[0276] In one embodiment, the method of preventing and / or managing adverse events comprises administering a “prophylactically effective amount” of tocilizumab, corticosteroid therapy, and / or an anti-epileptic drug for toxicity prophylaxis. In some embodiments, the method comprises administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, otilimab, mavrilimumab, a cytokine, and / or an anti-inflammatory agent.

[0277] In some embodiments, adverse events are managed by administering one or more agents that are antagonists or inhibitors of IL-6 or IL-6 receptor (IL-6R). In some embodiments, the agent is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds IL-6 or IL-6R. For example, in some embodiments, the agent is or includes tocilizumab (atlizumab) or sarilumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Patent 8,562,991. In some cases, the agent that targets IL-6 is an anti-TL-6 antibody, such as siltuximab, actoxumab, ALD518 / BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), and combinations thereof. In some embodiments, the agent can 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 an IL-6 mutein described in U.S. Patent 5591827. 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.

[0278] In some embodiments, other agents that can be used to manage adverse reactions and symptoms thereof include antagonists or inhibitors of cytokine receptors or cytokines. In some embodiments, the cytokine or receptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R / CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFa, TNFR1 such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-beta receptor (TGF-beta I, II, or III), IFN-gamma receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF alpha receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), IL-1 and IL-1R alpha / IL-1 beta. In some embodiments, the agent includes Simtuzumab, Sarilumab, Olokizumab (CDP6038), Actemra, ALD518 / BMS-945429, Sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the agent is an antagonist or inhibitor of a cytokine such as transforming growth factor 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 agent is an agent that targets a cytokine receptor (e.g., inhibits or is an antagonist of a cytokine receptor) such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-beta receptor (TGF-beta I, II, or III), IFN-gamma receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF alpha 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 prior to, after, or concurrently with the administration of the cells by one of the methods and dosages described elsewhere in this specification.

[0279] In some embodiments, the agent is administered at a dose of about 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg, 2 mg / kg to 4 mg / kg, or 6 mg / kg to 8 mg / kg, all inclusive, or at a dose of at least or at least about 2 mg / kg, 4 mg / kg, 6 mg / kg, or 8 mg / kg. In some embodiments, it is administered at a dose of about 1 mg / kg to 12 mg / kg, such as at or about 10 mg / kg. In some embodiments, the agent is administered by intravenous infusion. In one embodiment, the agent is tocilizumab. In some embodiments, the agent (e.g., specific tocilizumab) is administered prior to, after, or concurrently with the administration of the cells by one of the methods and doses described elsewhere in this specification.

[0280] In some embodiments, the method comprises identifying CRS based on clinical presentation. In some embodiments, the method comprises evaluating and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, CRS can be managed according to the recommendations in Protocol A, which can also be used in conjunction with other treatments of the present disclosure, including neutralization or reduction of the CSF / CSF R1 axis. Patients who develop > grade 2 CRS (e.g., hypotension, hypoxia that is unresponsive to fluid resuscitation, or requiring supplemental oxygen) should be monitored with continuous telemetry and pulse oximetry. In some embodiments, for patients who develop severe CRS, performing an echocardiogram to assess cardiac function is considered. For severe or life-threatening CRS, intensive care support therapy can be considered. In some embodiments, in the methods disclosed herein, a biosimilar or equivalent of tocilizumab can be used in place of tocilizumab. In other embodiments, another anti-IL6R can be used in place of tocilizumab.

[0281] In some embodiments, adverse events are managed according to the following protocol (Protocol A / Table 3):

[0282] Table 3: CRS grading and management guidelines

[0283]

[0284]

[0285] (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.

[0286] (b) For management of neurotoxicity, see Protocol B.

[0287] (c) For detailed information, see ACEMTRA ® Prescribing Information for Actemra® (tocilizumab), https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). The U.S. initial approval is noted as 2010.

[0288] (d) Alternative therapies include, but are not limited to: anakinra, siltuximab, ruxolitinib, cyclophosphamide, IVIG, and ATG.

[0289] Neurotoxicity

[0290] In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method comprises ruling out other etiologies for neurologic symptoms. Patients who develop > Grade 2 neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. Intensive support therapy is provided for severe or life-threatening neurotoxicity. Non-sedating anti-epileptic drugs (e.g., levetiracetam) are considered for seizure prophylaxis for any > Grade 2 neurotoxicity. The following treatments can be used in conjunction with other treatments of the present disclosure, including neutralization or reduction of the CSF / CSF R1 axis.

[0291] In some embodiments, NE is managed according to the following protocol (Protocol B / Table 4):

[0292] Table 4: Neurotoxicity grading and management guidelines

[0293]

[0294]

[0295] a Severity is based on the Common Terminology Criteria for Adverse Events grading.

[0296] b Alternative therapies include, but are not limited to: anakinra, siltuximab, ruxolitinib, cyclophosphamide, IVIG, and ATG.

[0297] Methylprednisolone can be substituted for equivalent levels of dexamethasone.

[0298] Additional safety management strategies with corticosteroids

[0299] Administration of corticosteroids and / or tocilizumab at Grade 1 can be considered prophylactic. Supportive care can be provided in all protocols at all CRS and NE severity levels.

[0300] In one embodiment of the regimen for managing adverse events related to CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only in the case of comorbidities or greater age); and / or corticosteroids (only in the case of comorbidities or greater age); Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids. In another embodiment of the regimen for managing adverse events related to CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: tocilizumab if no improvement after 3 days; and / or corticosteroids if no improvement after 3 days; Grade 2 CRS: tocilizumab; and / or corticosteroids; Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids, high dose.

[0301] In one embodiment of the regimen for managing adverse events related to NE, tocilizumab and / or corticosteroids are administered as follows: Grade 1 NE: no tocilizumab; no corticosteroids;

[0302] Grade 2 NE: no tocilizumab; no corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids (only if tocilizumab is not effective in improving); Grade 4 NE: tocilizumab; and / or corticosteroids.

[0303] In another embodiment of the regimen for managing adverse events related to NE, tocilizumab and / or corticosteroids are administered as follows: Grade 1 NE: no tocilizumab; and / or corticosteroids; Grade 2 NE: tocilizumab; and / or corticosteroids; Grade 3 NE: tocilizumab; and / or corticosteroids, high dose; Grade 4 NE: tocilizumab; and / or corticosteroids, high dose.

[0304] In one embodiment, corticosteroid treatment is initiated at CRS grade > 2 and tocilizumab treatment is initiated at CRS grade > 2. In one embodiment, corticosteroid treatment is initiated at CRS grade > 1 and tocilizumab treatment is initiated at CRS grade > 1. In one embodiment, corticosteroid treatment is initiated at NE grade > 3 and tocilizumab treatment is initiated at CRS grade > 3. In one embodiment, corticosteroid treatment is initiated at CRS grade > 1 and tocilizumab treatment is initiated at CRS grade > 2. In some embodiments, prophylactic use of tocilizumab administered on Day 2 can reduce the rate of > Grade 3 CRS.

[0305] In one embodiment, the regimen for treating adverse events includes Regimen C, as follows (Table 5).

[0306] Table 5: Alternate adverse event management guidelines

[0307]

[0308] a Tapering of treatment when symptoms improve at the discretion of the investigator;

[0309] b Not more than 800 mg; AE, adverse event; CRS, cytokine release syndrome; IV, intravenous; N / A, not applicable; NE, nervous system event

[0310] Any corticosteroid can be appropriate for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, both are administered. In some embodiments, the glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include, but are not limited to: alclometasone, alprogestone, beclometasone (e.g., beclometasone 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, deflazacort, desonide, desoximetasone, dexamethasone (e.g., dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodium phosphate), difluorosone (e.g., difluorosone diacetate), diflucortolone, difluprednate, glycyrrhizic acid, fluazacort, flucloronide, fluhydrocortisone (e.g., fluhydrocortisone acetate), flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, fluocortine, fluocortolone, fluorometholone (e.g., fluorometholone acetate), fluperolone (e.g., fluperolone acetate), fluprednisolone, fluprednipendil, flurandrenolide, fluticasone (e.g., fluticasone propionate), formocortal, halcinonide, halometasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone probutate, hydrocortisone buteate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), iclocimide, mazipredone, methasonone, methylprednisolone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), paramethasone (e.g., paramethasone acetate), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone phosphate sodium, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulphobenzoate, prednisolone tebutate, prednisolone tetrahydrophthalate), prednisone, prednisolone valerate, prednylidene, rimexolone, tixocortol, triamcinolone (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone 21-palmitate, triamcinolone diacetate).These glucocorticoids and salts thereof are discussed in detail in, for example, Remington's Pharmaceutical Sciences, A. Osol, Ed., Mack Pub. Co., Easton, Pa. (1980, 16th Ed.) and Remington: The Science and Practice of Pharmacy, 22nd Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2013), and any other editions, which are hereby incorporated by reference. In some embodiments, the glucocorticoid is selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a mineralocorticoid. Any other steroid can be used in the methods provided herein.

[0311] The one or more corticosteroids can be administered at any dose and frequency that can be adapted to the severity / grade of adverse events (e.g., CRS and NE). The above tables provide examples of dosing regimens for managing CRS and NE, respectively. In another embodiment, corticosteroid administration includes oral or IV administration of 10 mg dexamethasone 1 to 4 times per day. Another embodiment (sometimes referred to as a "high dose" corticosteroid) includes IV administration of 1 g methylprednisolone alone or in combination with dexamethasone per day. In some embodiments, the one or more corticosteroids are administered at a dose of 1 to 2 mg / kg per day.

[0312] Corticosteroids can be administered in any amount effective to ameliorate one or more symptoms associated with adverse events, such as CRS or neurotoxicity. For example, corticosteroids (e.g., glucocorticoids) can be administered to a 70 kg adult human subject in an amount of between at or about 0.1 and 100 mg, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to 40 mg, 0.1 to 30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg, 0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg, or 1 to 10 mg per dose. Typically, corticosteroids (such as glucocorticoids) are administered to an average adult human subject in an amount of between at or about 0.4 and 20 mg, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg per dose.

[0313] In some embodiments, a corticosteroid can be administered to an average adult human subject, typically weighing about 70 kg to 75 kg, for example, at or about a dose of 0.001 mg / kg (of the subject), 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.035 mg / kg, 0.04 mg / kg, 0.045 mg / kg, 0.05 mg / kg, 0.055 mg / kg, 0.06 mg / kg, 0.065 mg / kg, 0.07 mg / kg, 0.075 mg / kg, 0.08 mg / kg, 0.085 mg / kg, 0.09 mg / kg, 0.095 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.50 mg / kg, 0.55 mg / kg, 0.60 mg / kg, 0.65 mg / kg, 0.70 mg / kg, 0.75 mg / kg, 0.80 mg / kg, 0.85 mg / kg, 0.90 mg / kg, 0.95 mg / kg, 1 mg / kg, 1.05 mg / kg, 1.1 mg / kg, 1.15 mg / kg, 1.20 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.35 mg / kg, or 1.4 mg / kg.

[0314] Generally, the dose of corticosteroid administered depends on the particular corticosteroid, as there are differences in potency between different corticosteroids. It will generally be understood that drugs vary in their potencies, and therefore the dose will vary to achieve equivalent effects. The equivalence in potency of various glucocorticoids and routes of administration is well known. Information relating to equivalent steroid dosing (in a non-time dependent manner) can be found in the British National Formulary (BNF) 37, March 1999.

[0315] In some embodiments, adverse events are managed by the following regimen: patients receive levetiracetam (750 mg orally or intravenously, twice daily) starting on day 0 of T cell therapy administration; at onset of a Grade >2 neurological event, the levetiracetam dose is increased to 1000 mg twice daily; in one embodiment, if the patient does not experience any Grade >2 neurological events, the levetiracetam is tapered and discontinued as clinically indicated; patients also receive tocilizumab (8 mg / kg IV over 1 hour [not to exceed 800 mg]) on day 2; further tocilizumab (± corticosteroids) can be recommended at onset of Grade 2 CRS in patients with comorbidities or advanced age or in cases of Grade >3 CRS; for patients experiencing a Grade >2 neurological event, tocilizumab is initiated, and for patients with comorbidities or advanced age, or if there is any occurrence of a Grade >3 neurological event, corticosteroids are added if symptoms worsen despite use of tocilizumab. In some embodiments, if a neurological event occurs after prophylactic levetiracetam is stopped, levetiracetam is administered for prophylaxis at onset of Grade >2 neurotoxicity, and / or if the patient does not experience any Grade >2 neurotoxicity, levetiracetam is tapered and discontinued.

[0316] In some embodiments, adverse events are managed by the following regimen: patients receive dexamethasone 10 mg PO on day 0 (prior to T cell therapy infusion), day 1, and day 2; steroids are also administered starting at Grade 1 NE, and for Grade 1 CRS, no improvement is observed after 3 days of supportive care; if no improvement is observed after 24 hours of supportive care, tocilizumab is also administered for management of Grade >1 CRS.

[0317] Secondary malignancies

[0318] In some embodiments, patients receiving CAR T cell (e.g., CD19-directed) or other genetically modified autologous T cell immunotherapy treatment can develop secondary malignancies. In certain embodiments, patients receiving CAR T cell (e.g., CD19-directed) or other genetically modified allogeneic T cell immunotherapy treatment can develop secondary malignancies. In some embodiments, the method comprises lifelong monitoring for secondary malignancies.

[0319] Patents and scientific literature referred to herein establish the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference herein in their entirety. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, genomic database sequences, and scientific literature cited herein are hereby incorporated by reference. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, as to any definitions provided in any reference incorporated herein by reference, those definitions are hereby incorporated by reference herein, but only as of the filing date of this application, and any definition provided in this application controls in the event of an inconsistency with a contradictory definition provided in the reference.

[0320] The following examples are intended to illustrate various aspects of the present application. Accordingly, the particular aspects discussed are not to be interpreted as limiting the scope of the present application. For example, although the following examples are directed to T cells transduced with an anti-CD 19 chimeric antigen receptor (CAR), those skilled in the art will appreciate that the methods described herein can be applied to immune cells transduced with any CAR. The methods are also applicable to other immunotherapies. It will be apparent to those skilled in the art that various equivalents and modifications can be made without departing from the scope of the present application, and it is to be understood that such equivalents and modifications are to be included within the scope of the present application.

[0321] The disclosure provided herein can be used in methods other than those described above or as a combination of the methods described above. The following is a compilation of exemplary methods that can be derived from the disclosure provided herein.

[0322] Examples

[0323] Example 1

[0324] High B-cell signature correlates with improved EFS and higher probability of durable response after axi-cel treatment

[0325] To identify novel prognostic markers in LBCL, gene expression analysis was performed on pre-treatment tumor biopsy samples (collected at initial diagnosis or prior to lymphocyte depleting chemotherapy) and the NanoString PanCancer IO360 ™ Panel was used to evaluate pre-defined gene expression signatures (Table 6). In patients who received second line axi-cel treatment, the B-cell signature (IO360 ™) was the only pre-specified signature significantly associated with clinical benefit (P < 0.05) for all three efficacy endpoints: increased probability of ongoing response (vs. patients who progressed after response; improved EFS; and prolonged duration of response (DOR) (P < 0.05; patients with high signature values [> median] vs. low signature values [≤ median]). The pre-specified B cell lineage signature included BLK, CD19, MS4A1, TNFRSF17, FCRL2, FAM30A, PNOC, SPIB, and TCL1A. Of these genes, expression of CD19, MS4A1, and TNFRSF17 was significantly elevated in patients who were in ongoing response (descriptive P < 0.05), by 22%, 40%, and 69%, respectively. In contrast, expression of the hypoxia-associated gene, nitric oxide synthase 2 (NOS2), and the NK CD56dim NanoString signature (composed of IL21R, KIR2DL3, KIR3DL1, and KIR3DL2) was significantly associated with shorter EFS and / or DOR (P < 0.05).

[0326] In the SOC group, the B cell signature was not associated with efficacy, and few NanoString signatures were associated with efficacy endpoints. Immune gene expression signatures for macrophages, myeloid cells, antigen presentation machinery (APM), natural killer (NK) cells, or CD8 T cells were only weakly associated with one of the three efficacy endpoints (no signature was consistently associated with all three efficacy measures), suggesting that enrichment of specific tumor immune infiltrates can be one factor supporting SOC response. Nonetheless, EFS was superior for axi-cel vs. SOC in all subgroups, including the high APM (> median) subgroup.

[0327] Table 6: NanoString IO360 ™ panel gene signature

[0328]

[0329]

[0330]

[0331]

[0332] In the above table, genes that are negatively correlated with the cluster they are in are shown in bold. All other genes show positive correlation with the cluster. APM, antigen processing machinery; IFN, interferon; JAK, Janus kinase; MAGE, melanoma antigen gene; MHC, major histocompatibility complex; MMR, mismatch repair; MSI, microsatellite instability; NK, natural killer cell; STAT, signal transducer and activator of transcription; Thl, type 1 helper T cell; TIS, tumor inflammation signature; Treg, regulatory T cell.

[0333] Clustering analysis reveals distinct gene expression signatures, consistent with different TME immune contexts

[0334] NanoString IO360 ™ Unsupervised clustering analysis of the results identified four major gene expression signature clusters, reflecting different tumor microenvironment (TME) immune contexts (Table 6). The combination of features within each cluster was used to create indices for further analysis. The first cluster, referred to herein as the B-cell lineage and proliferation index (BPI), contained features such as B-cells, proliferation, APM deficiency, and glycolytic activity. The BPI signature was the most hierarchically separated from the other three major clusters, suggesting that this cluster represents a relatively simpler TME with abundant and highly proliferative cancer cells and lower immune cell infiltration compared to the other clusters. The second cluster, referred to as the stroma and immune suppression index (SII), contained a feature gene set including stromal cells, myeloid cells and endothelial cells, NOS2, transforming growth factor beta (TGF-b), B7-H3, arginase 1 (ARG1), and hypoxia. In this cluster, the hypoxia and NOS2 features (IO360) were negatively correlated with EFS and / or DOR following axi-cel treatment. The third cluster was enriched for features of NK cells, macrophages, and antigen presenting cells. The fourth cluster was primarily composed of T-cell infiltration features. The third and fourth clusters showed relatively close hierarchical correlation, possibly representing together tumors that are more complex and characterized by immune infiltration.

[0335] SII and BPI (TME context) correlate with EFS in the axi-cel group

[0336] Root mean square indices were generated for each of the above 4 NanoString signature clusters (see “Methods” section). BPI (cluster 1) and SII (cluster 2) were positively and negatively associated with EFS in the axi-cel group, respectively (descriptive P < 0.05). None of the 4 clusters were associated with cell of origin. Of note, BPI was positively associated with high grade B-cell lymphoma (HGBL) and double / triple hit disease. The median EFS for the HGBL subgroup after axi-cel treatment was 21.5 months (95% CI: 3.7 - not estimable; unstratified HR [axi-cel vs. SOC] = 0.318). While EFS was not significantly different between the HGBL subtype and the non-HGBL subtype (DLBCL+other), a more favorable direction of EFS was observed for the HGBL subtype vs. the non-HGBL subtype in the axi-cel group (unstratified HR [HGBL vs. non-HGBL] = 0.692; 95% CI: 0.384-1.245), suggesting that HGBL patients can benefit more from axi-cel than other patients. In contrast, the SOC group showed the opposite, non-statistically significant direction of association (unstratified HR [HGBL vs. non-HGBL] = 1.17; 95% CI: 0.723-1.892). The third and fourth clusters were not associated with EFS and / or DOR after axi-cel treatment. In contrast, none of the four clusters were significantly associated with outcomes in the SOC group, suggesting a difference in the impact of the TME on SOC and CAR T cell therapy outcomes.

[0337] Based on TME and therapy, CD19 expression level has differential impact on efficacy

[0338] CD19 protein levels (H-score) were associated with CD19 gene expression and B-cell signature. Consistent with the role of the B-cell signature in axi-cel-mediated efficacy, CD19 gene expression and protein levels were also associated with axi-cel EFS. Patients with higher CD19 gene and protein expression levels (> median) had superior axi-cel EFS relative to patients with lower expression levels (≤ median). Of note, axi-cel was superior to SOC in each CD19 gene expression subgroup. Patients who were CD19 negative by immunohistochemistry (H-score < 5) still demonstrated a significant response to axi-cel with an objective response rate (ORR) of 85% vs. 67% in the SOC group.

[0339] Patients with low CD19 protein expression (H-score ≤ median) exhibit a more complex TME with immune infiltration characteristics, rich in various immunosuppressive features, including regulatory T cells, T cell exhaustion, ARG1, indoleamine 2,3-dioxygenase 1 (IDO1), B7-H3, CTLA4, and gene expression signatures of macrophages and myeloid cells. The worst EFS was observed after axi-cel treatment in tumor patients with low CD19 protein expression and high SII, suggesting that both TME immunosuppression and target expression play a role in CAR T-cell therapy resistance. Conversely, in patients with high CD19 protein expression (> median), a lack of durable response was associated with glycolytic activity.

[0340] Axi-cel products enriched in CCR7+CD45RA+ T cells may overcome low antigen (CD19) expression.

[0341] Further research was conducted on CCR7+CD45RA+ T cells (considered to be naïve T cells or stem cell memory phenotypes (T cells)). SCM The question remains whether the axi-cel product can overcome the negative effects of adverse TME. Indeed, patients with relatively low CD19 expression showed improved EFS when the frequency of CCR7+CD45RA+ T cells in the product was high. Similar results were obtained with the total number of infused CCR7+CD45RA+ T cells. Patients with relatively high SII also showed improved EFS when the frequency of CCR7+CD45RA+ T cells in the infused product was high, but this difference was not statistically significant.

[0342] ZUMA-7 is the largest available clinical dataset in the field of second-line LBCL CAR T-cell therapy. This article explores the ZUMA-7 dataset to reveal novel tumor biomarkers associated with responses (EFS, DOR, sustained response, CR, objective response) to CAR T-cell therapy (axi-cel) or SOC (HDT-ASCT). The outcomes of axi-cel or SOC have been identified as being influenced by differential TME composition in second-line treatment, providing insights into the inferred mechanisms driving responsiveness to these therapies. For example, tumor gene expression characteristics, including B-cell scores and clusters rich in matrix and immunosuppressive features (SII), were positively and negatively correlated with CAR T-cell therapy outcomes, respectively. Notably, within the B-cell score, CD19 gene and protein expression were also positively correlated with cell therapy outcomes.

[0343] The analyses reported herein identified major clusters of gene expression signatures. SII, which was negatively associated with clinical outcome (cluster 2 in Table 6), can reflect an immunosuppressive TME, including myeloid-associated immunosuppression and TGF-b-activated stromal genes. Within this immunological context, CAR T cells can be unable to fully migrate to malignant cells and also maintain a functional state. In contrast and unexpectedly, BPI (cluster 1 in Table 6) was positively associated with HGBL / double-hit / three-hit disease and favorable clinical outcomes. This high-risk subgroup was more likely to have a high BPI signature, indicating a more homogenous population of malignant B cells and lower immune infiltrate diversity.

[0344] Another finding of this study was that there were clear distinctions between axi-cel biomarkers associated with outcome and SOC biomarkers. Interestingly, while the pre-specified B cell lineage signature and CD19 expression were positively associated with outcome following axi-cel treatment, other TME immune signatures, including APM and DC, were positively associated with outcome following SOC. This suggests that the mechanism of CAR direct engagement of CD19 antigen under axi-cel is different from the mechanism of mobilizing endogenous immune to fight tumor epitopes under SOC. Notably, axi-cel had superior outcomes than SOC in all presented biomarker subgroups.

[0345] In summary, these findings reveal novel gene expression signatures with potential predictive value for axi-cel response. These findings also help guide treatment decisions, including whether a patient would benefit from second-line cell therapy over standard of care. The four TME signature clusters described herein, as well as the association of cluster 1 (BPI) and cluster 2 (SII) with CAR T cell therapy outcomes, have not been previously reported.

[0346] Presented herein is a strong association between cluster 1 (B cell proliferation) and HGBL. Also reported herein is the association of the B cell proliferation cluster and the stromal cluster with best and worst outcomes, respectively. These observations can have important implications as CAR T cell therapy and other therapies move to earlier treatment lines, as these TME signatures can have predictive value for response.

[0347] This report presents previously unrecognized correlations between baseline CD19 expression and TME immune infiltration and with axi-cel outcomes. The association between CD19 expression and outcomes was discovered through IHC or NanoString gene expression profiling. Regardless of CD19 protein expression / H-score, axi-cel demonstrated superior EFS to SOC. However, in the axi-cel arm of ZUMA-7, patients with lower CD19 protein expression presented with a more complex, immune-infiltrated TME, which highlights that the relatively shorter EFS of axi-cel in patients with relatively lower CD19 protein expression (H-score < median) can depend not only on suboptimal target expression, but also on the concurrent presence of a confounding immune contexture feature. In fact, low CD19 H-score correlated with Cluster 2 (SII), which was negatively associated with axi-cel EFS, and the association between CD19 H-score and EFS appeared to be limited to patients with high SII index.

[0348] While patients with attenuated B-cell features and less favorable tumor immune microenvironments presented with a poorer clinical prognosis, a key question is whether modifiable product characteristics can help overcome such adverse features. As described herein, CAR T cell products enriched for the CCR7+CD45RA+ T cell phenotype can improve outcomes in patients with lower CD19 protein expression and higher immune suppressive features.

[0349] Understanding the immune contexture is critical to understanding the mechanism of action of CAR T cell therapy and the potential for durable responses. In addition to SPD, MTV, LDH, and target (CD19) expression, measuring tumor immune contexture using Immunoscore, IS21, B-cell, and stroma and immune suppressive gene signatures is emerging as important and interrelated determinants of durable responses to axi-cel intervention. The support for earlier CAR T cell therapy intervention in immune contextures more favorable to augment axi-cel activity can contribute to the more favorable efficacy of axi-cel compared to SOC across common prognostic subgroups of second-line LBCL. Furthermore, patients with HGBL / double hit / three hit disease, a feature associated with poorer outcomes to conventional chemoimmunotherapy, derived significant benefit from axi-cel treatment. The enrichment of BPI in HGBL / double hit / three hit tumors, in turn leading to higher prevalence of proliferative B-cells, CD19 expression, and relatively fewer immune suppressive cells, can highlight the sensitivity of this high-risk population to CAR T cell therapy targeting CD19 compared to SOC.

[0350] Patient samples and efficacy indicators

[0351] Evaluable samples from patients in the ZUMA-7 (N=170) and ZUMA-1 Cohort 1+2 (N=101) safety analysis sets were analyzed. The safety analysis set for ZUMA-7 was defined as patients who received at least one dose of axi-cel or SOC after randomization. The safety analysis set for ZUMA-1 was defined as all patients who received any dose of axi-cel treatment. These studies were approved by the institutional review boards of each study site and were conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice guidelines.

[0352] The efficacy endpoints (ORR, best response, EFS, DOR, and durable responses) for ZUMA-7 used the primary analysis data cutoff date. EFS was defined as the time from randomization to the earliest date of disease progression according to Lugano classification, initiation of a new lymphoma treatment, or death from any cause. Durable responses were defined as patients who were still in a state of ongoing response (CR or partial response (PR)) at the ZUMA-7 primary analysis data cutoff date. Post-response progression was defined as patients who achieved CR or PR and subsequently experienced disease progression. Patients who achieved stable disease or disease progression as best response were categorized as non-responders as previously reported. To place specific findings in context, data from patients with evaluable samples in ZUMA-1 Key Cohort 1+2 were included with a minimum follow-up of at least 60 months.

[0353] Analysis of gene expression signatures

[0354] ZUMA-1 and ZUMA-7 were similar in tumor biopsy collection and processing of formalin-fixed paraffin-embedded biopsy specimens. Gene expression and molecular subgroup analysis were performed using NanoString PanCancer IO360 ™ Panel and lymphoma subtype testing. Prespecified gene expression signatures from NanoString (proprietary algorithm) were analyzed for cluster analysis and association with efficacy endpoints. Non-supervised cluster analysis of gene expression signatures was performed using the computed hierarchical clustering method (UPGMA; distance metric - Euclidean, ordering weight - mean, null replacement method - constant value, replaced with 0 and not normalized) in TIBCO Spotfire. IS21 (a pre-specified T-cell infiltration and function gene expression signature) was computed using Veracyte’s proprietary algorithm based on gene expression values from the PanCancer IO360 ™ Panel. Cell subtypes within the TME and their association with clinical outcomes were investigated based on the PanCancer IO360 ™ Panel.

[0355] Analysis of CD19 expression level

[0356] CD19 protein expression levels were measured by IHC at NeoGenomics using a validated assay.

[0357] Analysis of product attributes

[0358] Product T cell phenotype and other product attributes were assessed as previously described. In the CellCarta lab, co-stimulatory (CD27, CD28) as well as activation and exhaustion markers (Programmed cell death protein 1, T cell immunoglobulin and mucin domain containing 3, Lymphocyte-activation gene 3) were additionally characterized by flow cytometry using validated assays.

[0359] Association analysis and correlation statistics

[0360] Associations between biomarkers from the exploratory endpoints and each other and with efficacy endpoints were analyzed. Spearman rank correlation analysis was used to assess associations between analytes. Kaplan-Meier curves and Cox regression were used to assess associations between biomarkers and time-to-event endpoints. Wilcoxon rank-sum test and logistic regression were used to assess relationships between biomarkers and binary outcomes. Kruskal-Wallis test was used to assess associations between biomarkers and categorical endpoints. For these post-hoc analyses, all P values are descriptive, and P < 0.05 was considered significant. No adjustment for multiple testing was made. Covariates were stratified into subgroups by median, quartiles, or specified values (e.g., SPD values 3721 mm 2 ) were used to generate plots using TIBCO Spotfire, SAS, R, or GraphPad Prism.

[0361] Example 2

[0362] This example demonstrates that, in the international phase 3 ZUMA-7 trial, axicel (axi-cel; autologous anti-CD19 chimeric antigen receptor T cell therapy) as second-line therapy significantly improved event-free survival for patients with early relapsed or refractory large B-cell lymphoma (R / R LBCL) compared to the standard-of-care group.

[0363] Patients were randomized in a 1 : 1 ratio to axi-cel or the standard-of-care group (2 to 3 cycles of a regimen-specified chemotherapy immuno-therapy followed by high-dose chemotherapy with autologous stem cell transplant [HDT-ASCT] for patients who responded). The primary analysis of overall survival in the intent-to-treat population was conducted per protocol 5 years after the first patient was randomized.

[0364] Overall, 359 patients were randomized to the axi-cel group (n=180) or the standard-of-care group (n=179). At a median follow-up of 47.2 months, axi-cel showed a statistically significant improvement in overall survival compared to standard-of-care (hazard ratio, 0.726; 95% CI: 0.540-0.977; stratified log-rank P=0.0168). Median overall survival was not reached in the axi-cel group and was 31.1 months in the standard-of-care group; estimated 48-month overall survival rates were 54.6% and 46.0%, respectively. The survival benefit of axi-cel was consistent across all pre-specified key subgroups, including: patients aged >65 years, and patients with primary refractory or high-grade B-cell lymphoma. Median investigator-assessed progression-free survival was 14.7 months in the axi-cel group and 3.7 months in the standard-of-care group, with estimated 48-month PFS rates of 41.8% and 24.4%, respectively (hazard ratio 0.506; 95% CI: 0.383-0.669). Axi-cel significantly improved overall survival as a second-line treatment for R / R LBCL compared to standard-of-care.

[0365] The standard of care for second-line treatment of cure-oriented large B-cell lymphoma (LBCL) for the past 30 years has been multiple courses of platinum-based chemotherapy followed by high-dose therapy with autologous stem cell transplant (HDT-ASCT) for patients who respond. However, only about half of patients are likely to be eligible for this regimen, and of those, only about 20% are ultimately cured. Patients who are unable to undergo HDT-ASCT have a poor outcome, with a median overall survival (OS) of 4.4 months.

[0366] Given this unmet need and the approval of chimeric antigen receptor (CAR) T-cell therapy in the third- or later-line setting, the phase 3 ZUMA-7 trial (NCT03391466) was designed to compare the efficacy of a single dose of anti-CD19 autologous CAR T-cell therapy axicel with second-line standard-of-care therapy in patients with early relapsed or primary refractory (R / R) LBCL. The primary endpoint, event-free survival (EFS), assessed by blinded central review, showed superiority of axicel over standard of care (hazard ratio [HR] 0.398; stratified log-rank test P < 0.0001). At a median follow-up of 24.9 months, the median EFS was 8.3 months versus 2.0 months, and the 24-month EFS rate was 41% versus 16% in the axicel and standard-of-care groups, respectively. Response rate, assessed by blinded central review, showed that 83% of patients in the axicel group and 50% of patients in the standard-of-care group had a response (complete response rate 65% versus 32%, respectively). We now report the primary OS analysis results of the ZUMA-7 trial at the protocol-specified time point of 5 years after the first patient was randomized.

[0367] Methods

[0368] Trial design and oversight

[0369] Adult patients (≥ 18 years of age) with histologically confirmed LBCL who were refractory to first-line treatment or who relapsed within 12 months after a complete response to first-line chemoimmunotherapy.

[0370] Patients were randomly assigned in a 1:1 ratio to the axicel group or the standard-of-care group (2 to 3 cycles of investigator-selected, protocol-specified chemoimmunotherapy followed by HDT-ASCT for patients who had a complete or partial response). Randomization was stratified by response to first-line therapy and by age-adjusted international prognostic index (aaIPI). Although treatment-group crossover was not planned in the protocol, patients could receive postprotocol follow-up therapy, including cellular immunotherapy after standard-of-care therapy (defined as a treatment switch).

[0371] Endpoints and assessments

[0372] The primary endpoint for ZUMA-7 was EFS (time from randomization to disease progression, death, or initiation of a new lymphoma therapy) as determined by blinded central review. Key secondary endpoints specified by the protocol included objective response rate and OS (time from randomization to death from any cause) as determined by blinded central review. Secondary endpoints included progression-free survival (PFS; time from randomization to disease progression or death from any cause) and EFS, reported here as investigator assessments because blinded central review was discontinued after the primary EFS analysis, per protocol. Disease assessments were performed at Day 50, Day 100, and Day 150 after randomization, and then every 3 months up to 2 years, followed by every 6 months up to 5 years of follow-up.

[0373] Statistical analysis

[0374] The planned primary OS analysis for the intent-to-treat (ITT) population was to be performed after 210 deaths were observed or no later than 5 years after the first patient was randomized, whichever came first. This analysis was triggered by the latter criterion. A group sequential testing procedure was performed on OS to control the overall one-sided alpha at 2.5%. The primary comparison of OS was performed using the log-rank test stratified by the randomization stratification factors with a power boundary one-sided significance level of 0.0249. In addition to the ITT analysis, two planned OS sensitivity analyses were performed to adjust for the confounding effect of switching (treatment crossover) from standard of care to protocol off-cell immunotherapy.

[0375] The efficacy analysis based on ITT principles included all randomized patients. The safety analysis included all randomized patients who received >1 dose of axi-cel or standard of care per protocol. All AEs from randomization to the visit at Day 150 after randomization or the change in lymphoma therapy, whichever came first, were reported. After Day 150, targeted serious AEs were reported to the data cutoff date, disease progression, or initiation of a new lymphoma therapy, whichever came first. Serious AEs assessed by the investigator as related to axi-cel were reported whenever they occurred.

[0376] Further methods

[0377] Study treatment

[0378] Patients in the axi-cel group received leukapheresis followed by cyclophosphamide (500 mg / m 6 / day) and fludarabine (30 mg / m 2 / day) on Days 5, 4, and 3 before a single infusion of axi-cel (target dose, 2 x 10 2Lymphodepleting chemotherapy was administered at a dose of 300 mg / m2of cyclophosphamide on days -6 and -5 and 24 mg / kg of fludarabine on days -6 to -3. Glucocorticoids were administered as an optional bridging therapy. Standard therapy group patients received 2 or 3 cycles of platinum-based chemotherapy as per institutional standard of care. Patients who had a complete response or partial response continued with high-dose chemotherapy with autologous stem cell transplant.

[0379] Endpoints and assessments

[0380] Event-free survival (EFS) was defined as the time from randomization to disease progression according to Lugano classification, initiation of new lymphoma therapy, death from any cause, or the earliest date of best response of stable disease through the assessment at day 150 after randomization, inclusive. Safety outcomes included incidence of adverse events (AEs). Management guidelines for CAR T cell-related AEs followed those used in ZUMA-1. AEs, including symptoms related to cytokine release syndrome (CRS) and neurologic events, were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. CRS severity was graded according to modified Lee criteria.

[0381] Reporting of serious adverse events

[0382] Targeted serious adverse events were defined as and included neurologic, hematologic, infectious, autoimmune, and secondary malignancy events, reported through 5 years for the standard therapy group or 15 years for the axi-cel group, or until disease progression, whichever occurred first. Serious adverse events assessed by the investigator as related to axi-cel were reported whenever they occurred.

[0383] Overall survival analysis

[0384] Overall survival (OS) was censored up to 3 times according to a group sequential testing procedure with a pre-specified rho family spending function controlling the overall one-sided alpha at 2.5%. According to the protocol, the first interim analysis of OS occurred at the time of the primary EFS analysis. The second interim analysis of OS was planned to occur after approximately 160 deaths were observed or no later than 4 years after the first patient was randomized. At the time of the primary EFS analysis, approximately 160 deaths were observed. Therefore, the interim analysis of OS at the time of the primary EFS analysis was the only interim analysis that met the criteria for both planned interim analyses of OS. The only subsequent planned analysis of OS was the primary OS analysis (reported herein) planned to occur after 210 deaths were observed or no later than 5 years after the first patient was randomized, which actually occurred 5 years after the first patient was randomized (because fewer than 210 deaths were observed at the protocol-specified time point).

[0385] Exploratory analyses

[0386] Exploratory analyses were performed to determine associations between OS and axi-cel pharmacokinetics and product characteristics. Anti-CD19 CAR T cell levels in blood were quantified as previously described, and product T cell phenotype and other attributes were assessed. Variables were characterized using the median value (i.e., < median and > median) as a cutoff for comparison.

[0387] In the axi-cel group, the presence of B cells in blood was assessed using flow cytometry as previously described up to 24 months. B cell aplasia was defined as no detectable B cell levels (i.e., below the lower limit of quantification of the assay used [<0.017% of white blood cells]). B cell recovery was defined as a detectable B cell level compared to a prior time point.

[0388] Persistent cytopenias were evaluated post-hoc over 4 time intervals and were defined as cytopenias that persisted beyond 6 months, 12 months, 18 months, and 24 months from the day after or following radical treatment initiation (i.e., from the time point of receiving axi-cel infusion or first high-dose treatment) according to the protocol.

[0389] Additional statistical methods

[0390] Kaplan-Meier estimates were provided for time-to-event endpoints as previously reported. Estimated hazard ratios and their two-sided 95% confidence intervals were calculated from Cox proportional hazards models stratified by the randomization stratification factors. Stratified log-rank P values (one-sided) were calculated for time-to-event endpoints.

[0391] To adjust for the confounding effect of switching from standard-of-care to off-protocol cellular immunotherapy (treatment switch) in the standard-of-care group, two validation methods were used for the pre-specified OS sensitivity analyses: a rank-preserving structural failure time (RPSFT) model with g-estimation and inverse probability of censoring weighting (IPCW) method. Patients in the standard-of-care group who received cellular immunotherapy were not censored at the cellular immunotherapy time point. In this model, the survival / death time after cellular immunotherapy for patients in the standard-of-care group was shortened as if the cellular immunotherapy was not given.

[0392] Associations between product characteristics or CAR T cell levels and efficacy endpoints were explored by post-hoc univariate analyses, and descriptive P values were reported. No adjustment for multiplicity testing was made. Covariates were continuous or categorized using the median. The percent increase in risk for each one-unit increase in a continuous variable was calculated as the hazard ratio by Cox regression models. Stratified (derived) Cox proportional hazards P values were calculated.

[0393] Results

[0394] Patients

[0395] Between January 25, 2018 and October 4, 2019, a total of 359 patients were enrolled and randomly assigned to the axi-cel group (n = 180) or the standard-of-care group (n = 179). Baseline demographics and disease characteristics were similar between treatment groups and were consistent with real-world patient populations receiving CAR T cell therapy (Table 7). High-risk features were prevalent, including 19% with high-grade B-cell lymphoma (HGBL; including double-hit lymphoma), 45% with high second-line aaIPI score (2 or 3 points), 54% with elevated lactate dehydrogenase levels, and 74% refractory to first-line therapy.

[0396] Table 7. Zuma-7 study participant demographics

[0397]

[0398]

[0399] Efficacy

[0400] At a median follow-up time of 47.2 months (range: 39.8-60.0), 82 patients died in the axi-cel group and 95 patients died in the standard-of-care group. The primary analysis of OS demonstrated a statistically significant improvement in OS with axi-cel compared with standard of care (death HR, 0.726; 95% CI: 0.540-0.977; stratified log-rank P = 0.0168). The median OS was not reached in the axi-cel group (95% CI: 28.6 months - not estimable [NE]), and was 31.1 months (95% CI: 17.1-NE) in the standard-of-care group (Table 8). The estimated 48-month OS rate was 54.6% (95% CI: 47.0-61.6) in the axi-cel group and 46.0% (95% CI: 38.4-53.2) in the standard-of-care group (Table 9). The OS benefit with axi-cel compared with standard of care was consistent across key pre-specified high-risk subgroups, including age ≥ 65 years, primary refractory disease, high second-line aaIPI score, and HGBL (including double-hit lymphoma) (Table 10).

[0401] Table 8. Overall survival. Kaplan-Meier estimates of overall survival. Data for patients not meeting event criteria were censored. One-sided P-values from log-rank tests are reported. Axi-cel, axicabtagene ciloleucel; HR, hazard ratio; NE, not estimable; NR, not reached; OS, overall survival Table 9. Kaplan-Meier estimates of overall survival for the axi-cel group and the standard of care group Table 10. .

[0402]

[0403] Overall survival by pre-specified subgroups. Subgroup analyses of overall survival by key baseline and clinical covariates, including response to first-line therapy and age-adjusted IPI at randomization. Hazard ratios and two-sided 95% confidence intervals for the estimate of the hazard ratio of axi-cel versus standard of care were provided using stratified Cox regression models. Conclusions of the Cox regression models were handled using the Breslow method. Axi-cel, axicabtagene ciloleucel; BCL, B-cell lymphoma; DLBCL, diffuse large B-cell lymphoma; HGBL, high-grade B-cell lymphoma; IPI, international prognostic index; LCI, lower confidence interval; UCI, upper confidence interval

[0404]

[0405] Table 11. Subsequent therapy received by drug class Table 12. Pre-specified overall survival sensitivity analysis adjusting for the impact of subsequent cellular immunotherapy in the standard of care group using a rank-restricted frailty time model. Kaplan-Meier estimates of overall survival from the sensitivity analysis using the rank-restricted frailty time method, which was designed to address the confounding effect of treatment switching in the standard of care group. Treatment switching rate was defined as the proportion of patients randomized to the standard of care who received a commercially available or investigational cellular immunotherapy after failing to respond to the standard of care or after relapse following the standard of care. One-sided P-values from log-rank tests are reported. Axi-cel, axicabtagene ciloleucel; HR, hazard ratio; NE, not estimable; NR, not reached; OS, overall survival ​ ​ ​ ​ .

[0406]

[0407]

[0408] In the standard-of-care group, 102 (57%) patients received subsequent cellular immunotherapy outside protocol due to disease progression or lack of response (Table 11). A pre-specified sensitivity analysis designed to assess the confounding effect of treatment crossover on OS in the standard-of-care group showed a more pronounced OS benefit with axi-cel compared to standard of care (Table 12).

[0409] ​ .

[0410]

[0411] ​ ​ ​ ​ ​ cel, axicel; HR, hazard ratio; NE, not estimable; NR, not reached; OS, overall survival.

[0412]

[0413] Investigator-assessed PFS confirmed the benefit of axi-cel compared to standard of care, with a median PFS of 14.7 months (95% CI: 5.4-43.5) in the axi-cel group and 3.7 months (95% CI: 2.9-5.3) in the standard-of-care group (HR, 0.529 [95% CI: 0.383-0.669]; descriptive one-sided P < 0.0001) (Table 13). The estimated 48-month PFS rate was 41.8% (95% CI: 34.1-49.2) in the axi-cel group and 24.4% (95% CI: 17.2-32.2) in the standard-of-care group. Investigator-assessed median EFS (different from the primary endpoint EFS determined by central review), was 10.8 months (95% CI: 5.0-25.5) in the axi-cel group and 2.3 months (95% CI: 1.7-3.1) in the standard-of-care group, with estimated 48-month EFS rates of 38.9% and 17.3%, respectively (HR, 0.422 [95% CI: 0.327-0.545]; descriptive one-sided P < 0.0001) (Table 14).

[0414] Table 13 and Table 14 below show investigator-assessed progression-free survival and event-free survival. Table 13 shows Kaplan-Meier estimates of investigator-assessed progression-free survival, defined as the time from randomization to the date of disease progression or death from any cause, as assessed by the investigator. Table 14 shows Kaplan-Meier estimates of investigator-assessed EFS, defined as the time from randomization to the earliest date of disease progression according to Lugano classification, initiation of a new lymphoma therapy, or death from any cause. For Tables 13 and 14, data for patients who did not meet the event criteria were censored. Stratified Cox regression models were used to provide hazard ratio estimates and two-sided 95% confidence intervals for the hazard ratio of axi-cel versus standard of care. Breslow methods were used to handle ties in the Cox regression model. One-sided P values from log-rank tests were reported. Axi-cel, axicabtagene ciloleucel; EFS, event-free survival; HR, hazard ratio; PFS, progression-free survival.

[0415] Table 13

[0416]

[0417] Table 14

[0418]

[0419] Safety

[0420] The safety analysis set included 170 patients who received axi-cel and 168 patients who received standard of care. All patients reported >1 treatment-emergent AE (TEAE); the cumulative incidence of any-grade TEAEs and >3-grade TEAEs, and serious TEAEs are shown in Table 15 and Table 16, respectively. In the safety analysis set, 74 patients in the axi-cel group and 91 patients in the standard of care group died after study start.

[0421] Table 15. Most common adverse events, cytokine release syndrome, and nervous system events .

[0422]

[0423]

[0424] Table 16. Serious adverse events reported by at least 3 patients in any treatment group .

[0425]

[0426]

[0427] Disease progression was the most common cause of death in the axi-cel group (n=51) and the standard-of-care group (n=71) (Table 17). Mortality related to curative treatment (axi-cel or HDT-ASCT) was 1 / 170 (1%) in the axi-cel group and 2 / 64 (3%) in the standard-of-care group (Table 17). Cumulative treatment-related serious AEs or fatal AEs have not changed since the primary EFS analysis.

[0428] Table 17. Cumulative deaths of treated patients in the axi-cel group and the standard of care group (safety analysis set) .

[0429]

[0430]

[0431] Since the start of the study, 11 patients have been reported to develop a new or secondary malignancy (8 patients treated with axi-cel, all assessed by the investigator as unrelated to axi-cel; 3 patients treated with standard of care, including 1 patient who developed 2 new malignancies) (Table 18). No cases of replication-competent retroviral infection have been reported.

[0432] Table 18. List of new secondary malignancies

[0433]

[0434]

[0435] In the axi-cel group, 76 patients (45%) and 28 patients (16%) reported any-grade and >3-grade infections, respectively, compared with 53 patients (32%) and 20 patients (12%) in the standard-of-care group (Table 19). Among the 162 axi-cel-treated patients who had B-cell level assessments, 47% had B-cell aplasia (undetectable B cells) within 3 months of infusion (Table 20; Table 21).

[0436] Table 19. Infections occurring during treatment by preferred term and grade .

[0437]

[0438]

[0439] Table 20. Summary of B-cell aplasia by baseline status .

[0440]

[0441]

[0442] Table 21. CAR T cell and B-cell levels over time. In evaluable patients, the percentage of B cells out of total white blood cells (median log-transformed ± 95% CI) and the number of anti-CD19 CAR T cells in the blood from baseline through 24 months after axi-cel infusion. Axi-cel, axicel; BLQ, below the limit of quantification; CAR, chimeric antigen receptor Table 22. Hypogammaglobulinemia occurring during treatment by most severe grade and IVIG useTable 23. Summary of cytopenias persisting 6, 12, 18, and 24 months or later after start of radical treatment by study protocol Exploratory transformation analysis .

[0443]

[0444]

[0445] Over time, B-cell recovery (i.e., an increase in the level of detectable B cells compared to a prior time point) was observed, although with substantial variability among patients. Median B-cell levels remained at or below the lower limit of quantification (0.017%) through 6 months post infusion, and increased starting at 9 months, a time point that coincided with the disappearance or reduction to very low levels of CAR T cells in the blood (median approximately equal to or below 0.1 cell / µL; Table 21, see above).

[0446] Hypogammaglobulinemia was reported in 11% and 1% of patients in the axi-cel and standard-of-care groups, respectively; all cases were Grade 1 or 2 (Table 22). In the axi-cel group, 28 patients (16%) received intravenous immunoglobulin therapy according to investigator judgment (Table 22).

[0447] Table 24. Overall survival by median peak anti-CD19 CAR T cell level in the blood (safety analysis set) .

[0448]

[0449] At or after 6 months after start of definitive therapy, 8 (5%), 0 (0%), and 6 (4%) patients in the axi-cel group (N=170) and 1 (2%), 1 (2%), and 0 (0%) in the standard-of-care group (n=62) were reported to have Grade ≥3 persistent cytopenias, thrombocytopenia, and neutropenia, respectively (Table 23).

[0450] Table 25. Overall survival by median AUC of anti-CD19 CAR T cell level in the blood (safety analysis set) Table 26. Association between axi-cel product characteristics and overall survival by time of event occurrence .

[0451]

[0452] Since the primary EFS analysis, no new CRS or neurologic events have been reported in either treatment group (Table 15, see above).

[0453] Table 27. Axi-Cel product characteristics

[0454] CAR T cell peak levels and area under the curve within the first 28 days post infusion were not significantly associated with OS (Tables 24 and 25). In axi-cel-treated patients, OS benefit was independent of axi-cel product characteristics, with two notable exceptions (Table 26).

[0455] Tables 24 and 25 show the association between overall survival and CAR T cell expansion. Kaplan-Meier estimates and estimated hazard ratios for overall survival in axi-cel patients, grouped by peak CAR T cell level (defined as the maximum number of CAR T cells in the blood after axi-cel infusion) after infusion (Table 24) and AUCo-28(Table 25). CAR T cell expansion was divided into subgroups based on the median. Using a stratified Cox regression model, the estimated hazard ratio and two-sided 95% confidence interval for axi-cel peak > median versus peak < median were calculated. Conclusions from the Cox regression model were handled using the Breslow method. One-sided P values from the log-rank test are presented. AUCo-28, area under the curve from day 0 to day 28; axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; HR, hazard ratio; OS, overall survival.

[0456] Further results Results of preplanned interim OS analysis

[0457]

[0458] Prespecified sensitivity analysis for OS Additional response outcomes

[0459]

[0460] Additional safety outcomes .

[0461]

[0462]

[0463]

[0464] In the OS versus product characteristic analysis, a higher proportion of naive or stem cell memory T cell phenotype (CCR7+CD45RA+ T cells) in the axi-cel product was associated with improved OS (stratified descriptive P = 0.0085) (not shown). Conversely, a higher proportion of differentiated T cells, particularly effector memory type (CCR7-CD45RA-), in the axi-cel product was associated with worsened OS (stratified descriptive P = 0.0091) (not shown). Table 27 below shows various product characteristics of the axi-cel product. More specifically, in comparison to the median value of the percentage of naive or stem cell memory T cells in the axi-cel product, patients with an elevated percentage of naive or stem cell memory T cell phenotype (i.e., naive T cells) were observed to have an improved overall survival, while in comparison to the median value of the percentage of differentiated T cells in the axi-cel product, patients with an elevated percentage of differentiated T cells were observed to have a decreased overall survival.

[0465] Discussion

[0466]

[0467]

[0468] Example 3

[0469] Table 28: Sample size and incidence of outcomes

[0470] As previously reported, a preplanned interim analysis of OS was conducted when the primary EFS analysis was conducted in ZUMA-7. In the intent-to-treat analysis population, the median OS was not reached for the axi-cel group and was 35.1 months for the standard-of-care group (hazard ratio [HR], 0.73; 95% CI: 0.53-1.01), with results favoring axi-cel. The interim analysis of OS was updated after additional survival follow-up of patients who discontinued treatment, as required by the U.S. Food and Drug Administration, incorporating information from public records through the data cutoff date of March 18, 2021. In this analysis, the median OS was not reached for the axi-cel group and was 25.7 months for the standard-of-care group (HR, 0.71; 95% CI: 0.52-0.97).

[0471] Table 29: Best data-driven model to predict grade >3 neurotoxicity

[0472] In a pre-specified sensitivity analysis designed to assess the confounding effect of treatment crossover on OS in the standard-of-care group, using a rank -restricted frailty time model analysis, axi-cel showed a significantly longer median OS than standard of care (not reached [95% CI: 28.6 months - unable to estimate] versus 15.5 months [95% CI: 9.7 - unable to estimate]; HR, 0.608 [95% CI: 0.449-0.824]; stratified log-rank one-sided descriptive P = 0.0006) (Table 12). Similar results were obtained using inverse probability of censoring weighting (HR, 0.633; 95% CI: 0.438-1.118).

[0473] Table 30: Best data-driven model to predict low-grade toxicity or no toxicity

[0474] In the current analysis, the investigator-assessed objective response rate was 83% (complete response rate 61%) in the axi-cel group and 45% (complete response rate 34%) in the standard-of-care group. The median duration of response was 41.7 months (95% CI: 13.6 - not estimable) and 7.8 months (95% CI: 5.0 - not estimable) in the axi-cel and standard-of-care groups, respectively. At the time of data cutoff, 71 / 180 (39%) and 29 / 179 (16%) patients in the axi-cel and standard-of-care groups, respectively, were in ongoing response.

[0475] Example 4

[0476] At the time of prior analysis, there were 2 patients with ongoing neurologic events: 1 patient randomized to the axi-cel group had grade 2 paresthesia and grade 1 memory disorder, both of which persisted until the patient’s death; 1 patient randomized to the standard-of-care group had grade 1 paresthesia, which was still ongoing at the time of long-term follow-up.

[0477] Introduction

[0478] In this trial, for patients with R / R LBCL, axi-cel treatment compared with standard-of-care treatment significantly reduced the risk of death by 27.4% and improved the absolute 4-year survival rate by 8.6% for the two second-line treatment strategies compared.

[0479] Prior to the advent of CAR T cell therapy, second-line LBCL patients who were ineligible for HDT-ASCT curative treatment had poor outcomes with a median OS of only 4.4 months. Prior attempts to improve survival in the second-line curative treatment setting have not been successful, suggesting that chemotherapy-based approaches have reached an upper limit of benefit; the most recent trial showing improved survival was the Parma study in 1995, which was conducted prior to the approval of rituximab. Thus, there is an unmet need for a new non-chemotherapy-based second-line treatment for high-risk patients with early-stage R / R LBCL, which drove the design of the ZUMA 7 study.

[0480] Based on the superiority of axi-cel versus chemotherapy / HDT-ASCT in EFS and response in the ZUMA-7 study, axi-cel became the first CAR T cell therapy approved for second-line treatment of LBCL in the United States, the European Union, and many other countries. The primary OS analysis results reported here ultimately confirmed a clear survival advantage for patients treated with axi-cel compared with patients treated with second-line platinum-based chemotherapy with (if they responded) HDT-ASCT. Importantly, the stability of the Kaplan-Meier survival curves for OS and PFS over 4 years indicates that second-line axi-cel treatment has the potential to cure a substantial proportion of patients.

[0481] The OS benefit of axi-cel versus chemotherapy / HDT ASCT was consistent across key prespecified patient subgroups with known poor prognosis, including patients aged ≥65 years, patients refractory to first-line therapy, and patients with HGBL. The benefit of axi-cel in older patients is particularly noteworthy because those who would otherwise be considered ineligible for curative HDT-ASCT treatment due to advanced age can still be eligible for CAR T cell therapy. Given the reported OS and EFS improvements with axi-cel in older patients that are at least as similar to the overall population, axi-cel can expand the population of patients who can benefit from curative therapy.

[0482] In addition, axi-cel was associated with a significant OS benefit despite more than half of the patients in the standard-of-care group receiving subsequent cellular immunotherapy outside of protocol due to lack of response or disease progression. Notably, this proportion is similar to other contemporary randomized anti-CD19 CAR T cell therapy trials that included a crossover design as part of the protocol. Historically, patients in the standard-of-care group who had early relapse or prior rituximab treatment had estimated 3-year OS rates of 39% and 40%, respectively, but patients with both early relapse and prior rituximab treatment (representing the ZUMA 7 study patient population) had a 3-year survival rate of less than 40%. In contrast, the estimated 3-year OS rate for patients in the standard-of-care group in the ZUMA-7 study was 48%. Given that survival in the standard-of-care group was improved compared with historical studies during the conduct of the ZUMA-7 study due to the availability of a third-line CAR T cell therapy, the true survival benefit of second-line axi-cel compared with standard of care can be greater, which is supported by our treatment crossover analysis. Based on the clear improvement in survival with axi-cel compared with platinum-based chemotherapy / HDT-ASCT, axi-cel should be the standard of care for second line, rather than attempting second-line chemotherapy and administering cellular immunotherapy only after demonstrating a lack of adequate response.

[0483] Long-term safety of axi-cel was consistent with prior studies. Notably, the AE reporting period ended upon disease progression or initiation of new lymphoma treatment, both of which occurred at a significantly higher rate in the standard-of-care group. Persistent cytopenias and immune deficiencies, including induction of B-cell aplasia and infections, are expected with CD 19 -targeted CAR T cell therapy, representing a target-related / non-tumor class effect. Notably, the incidence of persistent grade >3 cytopenias decreased over time, with a decline starting at 6 months post-axi-cel infusion. Given the incidence of grade >3 infections, hypogammaglobulinemia, and B-cell aplasia with axi-cel treatment, clinical monitoring of patients treated with CAR T cell therapy is important to reduce the risk of long-term infections. B-cell recovery was observed over time in the majority of patients treated with axi-cel, suggesting that durable clinical benefit does not depend on long-term persistence of functional CAR T cells, as previously described with axi-cel in third- or later-line treatment.

[0484] In addition to improving survival, axi-cel was associated with improved quality of life (QoL). Compared with standard-of-care, patients treated with axi-cel had significantly longer time to clinical symptoms or toxicity, gained a clinically important gain in quality-adjusted survival, had a clinically meaningful improvement in QoL, and returned to baseline levels more quickly.

[0485] Importantly, OS is an objective endpoint that is not subject to observer bias, and the primary OS analysis from ZUMA-7 ultimately established the following conclusion: for early R / R LBCL, a second-line axi-cel treatment strategy is superior to chemotherapy followed by HDT-ASCT in patients who respond to the chemotherapy. When platinum-based chemotherapy is used as the initial second-line treatment, only a small number of patients receive curative HDT-ASCT, primarily due to lack of chemotherapy sensitivity (which is unknown at the time treatment is initiated). The OS improvement with axi-cel highlights the importance of early referral for axi-cel treatment prior to initiating second-line chemotherapy.

[0486] The ZUMA-7 study demonstrated that axi-cel significantly improved survival compared with second-line chemotherapy / HDT-ASCT in patients with R / R LBCL and that long-term outcomes were consistent with curative therapy.

[0487] Methods

[0488] This example discloses a classification modeling method for identifying the risk of developing neurotoxicity and CRS in second-line R / R LBCL patients upon receiving axi-cel treatment.

[0489] axi-cel is an autologous anti-CD 19 CAR T cell therapy approved for the treatment of relapsed / refractory (R / R) large B-cell lymphoma (LBCL) after ≥1 lines of systemic therapy. In the randomized Phase 3 ZUMA-7 trial, it showed efficacy superior to standard of care in the second-line (refractory or early relapsed) setting. After treatment with axi-cel, 92% and 61% of patients reported cytokine release syndrome (CRS) and neurologic events (NEs), respectively; the incidence of high-grade (> Grade 3) events was 6% and 21%, respectively. The pathophysiology of CRS is well documented, whereas the underlying mechanisms of NEs remain unclear. Prospective identification of patients at low risk of toxicity (who can be treated in an outpatient setting) is of practical interest.

[0490] Here, the goal is to create a data-driven multivariate classification model for second-line R / R LBCL using pre-treatment biomarkers to identify patients with the following toxicity-related outcomes: Grade 3+ neurologic events (NEs) at any time after axi-cel treatment, and low-grade toxicity or no toxicity (CRS ≤ Grade 1 at maximum within 7 days after axi-cel treatment and no NEs). We also sought to validate the model that performed best in multiple R / R LBCL populations as shown in Table 28 below.

[0491] We included approximately 450 biomarker + timepoint combinations. Candidate biomarkers included: demographic indicators (approximately 5), clinical and disease characteristics (approximately 30), routine serum chemistry and hematology indicators (approximately 30), cytokine and other pharmacodynamic markers (approximately 25), and product attributes (approximately 30). Timepoints at which these biomarkers were measured included: baseline (i.e., prior to lymphodepletion), Day 0 (prior to axi-cel treatment); fold change from baseline to Day 0 was also considered, calculated as (Day 0 value) / (baseline value). Machine learning methods (e.g., conditional random forests, XGBoost) were used for feature selection. Selected features were used to train logistic classifiers, and their performance was evaluated in a held-out test set (70%:30% split). Bootstrap resampling was used to estimate variability. A data-driven approach was taken (i.e., no pre-selected set of putative clinically relevant biomarkers). Clinician-curated models were selected from the top data-driven models.

[0492] Patients and study design

[0493]

[0494] Table 29 shows the best model results identified using data-driven methods for predicting Grade >3 neurotoxicity. Model selection was based on high area under the receiver operating characteristic curve (AUROC), high negative predictive value (NPV), and consistent covariate directionality across training and test sets. In Table 29, the higher the biomarker level, the higher the likelihood of Grade >3 neurotoxicity.

[0495] Procedures and endpoints

[0496]

[0497] Table 30 shows the results of top data-driven models for low-grade or no toxicity. Model selection was based on high AUROC, high positive predictive value (PPV), and consistent covariate directionality across training and test sets. In Table 30, the higher the biomarker level, the higher the likelihood of low toxicity or no toxicity.

[0498] Statistical analysis

[0499]

[0500] While it remains challenging to predict which patients will experience high-grade NE toxicity, the results of the present study suggest good performance (i.e., high NPV) in prospectively identifying a subset of patients unlikely to experience high-grade NE toxicity. The results disclosed herein include a classifier that performed best for low-grade toxicity or no toxicity (defined as CRS < Grade 1 and no NE), with a PPV of 70% (7 out of 10 correct classifications) in both the training and test sets, suggesting it can be a pathway to identify patients suitable for outpatient dosing. This modeling effort can help support clinicians in prospectively managing patients at risk for toxicity, as well as those who can be suitable for outpatient monitoring.

[0501] Results

[0502] Axi-cel, an autologous anti-CD19 CAR T-cell therapy, is approved for the treatment of relapsed / refractory (R / R) follicular lymphoma (FL). This approval was supported by the phase 2, multicenter, single-arm ZUMA-5 study evaluating the efficacy of axi-cel in patients with R / R indolent non-Hodgkin lymphoma (iNHL; N=104), including FL and marginal zone lymphoma [MZL]. In the primary analysis (median follow-up 17.5 months), the overall response rate (ORR) was 92% (complete response rate 74%). Here, we report long-term results from ZUMA-5. Eligible R / R iNHL patients after ≥2 lines of therapy underwent leukoablation followed by lymphocyte depletion chemotherapy and axi-cel infusion (2 × 10⁻⁶). 6 CAR T cells / kg. The primary endpoint was ORR, which was assessed by the investigator in all enrolled patients (intention-to-treat population) in this analysis. After a median follow-up of 41.7 months in FL patients (n=127) and 31.8 months in MZL patients (n=31), the ORR was comparable to the primary analysis (94% for FL; 77% for MZL). The median progression-free survival was 40.2 months in FL patients, and the median progression-free survival for MZL patients has not yet been reached. Median overall survival was not reached in either disease type. Grade ≥3 concern adverse events since the last analysis were primarily seen in patients who had recently received treatment. Clinical and pharmacokinetic outcomes were negatively correlated with recent bendamustine exposure and high-metabolic tumor volume. After 3 years of follow-up in the ZUMA-5 study, axi-cel demonstrated a sustained and durable response in R / R iNHL patients with very low relapse rates after 2 years and a manageable safety profile.

[0503] Patients

[0504] Relapsed or refractory (R / R) indolent non-Hodgkin lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL), is considered essentially incurable, and most patients eventually experience additional disease relapse. In FL patients, second-line treatment is heterogeneous, but a common thread is the progressively shorter remission period following second-line therapy and subsequent treatments, leading to shorter survival. Furthermore, FL patients who progress within 24 months of initiating their first anti-CD20 chemoimmunotherapy (POD24) have a poor prognosis and shorter survival with existing R / R treatment regimens.

[0505] Recent advances in the treatment options for iNHL, including chimeric antigen receptor (CAR) T-cell therapy, have improved outcomes for patients with R / R disease. Axi-cel is an autologous anti-CD19 CAR T-cell therapy that contains a CD28 costimulatory domain to elicit rapid and robust expansion, leading to target-specific cytotoxicity and helping to overcome the limitations of the immune system. Axi-cel was approved for the treatment of adult patients with R / R FL. This approval was supported by the primary analysis of the ZUMA-5 trial, a single-arm, international phase 2 study in patients (N = 104) with iNHL, who had a median follow-up of 17.5 months and an overall response rate (ORR) of 92% (complete response [CR] rate 74%).

[0506] Because of the heterogeneity of pre-treatment tumor characteristics and their protracted clinical course, long-term follow-up analyses are particularly important in R / R indolent lymphoma. This report presents efficacy, safety, and biomarker assessment results from the ZUMA-5 study at 3 years of follow-up, the longest follow-up analysis to date for CAR T-cell therapy in iNHL. This analysis included an exploratory assessment of the association of clinical outcomes with baseline variables, including prior bendamustine exposure and baseline tumor burden assessed by metabolic tumor volume (MTV).

[0507] Table 31. Baseline characteristics of follicular lymphoma patients by prior bendamustine use (before and after propensity score matching)

[0508] Efficacy in follicular lymphoma patients

[0509] ZUMA-5 was a multicenter, single-arm, registrational phase 2 trial conducted at 17 medical centers in the United States and France, registered at Clinicaltrials.gov / NCT03105336. The complete list of sites has been previously reported. All enrolled patients signed a written informed consent form to participate in the study, and the study protocol was approved by the institutional review board at each study center.

[0510] The complete patient eligibility criteria have been previously reported. Briefly, patients aged ≥18 years with R / R iNHL, including FL (grades 1-3a) and MZL (nodal or extranodal; both per WHO 2016 criteria), who had received ≥2 lines of systemic therapy, including one anti-CD20 monoclonal antibody in combination with one alkylating agent, were enrolled. Patients who had received an autologous stem cell transplant (SCT) within 6 weeks before axi-cel infusion, received any allogeneic SCT, CD19-targeted therapy, or CAR T-cell therapy were excluded. Disease progression within <6 months of completing the most recent prior therapy was considered refractory disease.

[0511] Table 32. PFS of follicular lymphoma patients by time point of bendamustine use prior to leukapheresis

[0512] Enrolled patients underwent leukapheresis followed by lymphodepleting chemotherapy with fludarabine (30 mg / m 2 / day) and cyclophosphamide (500 mg / m 2 / day) on days -5 to -3 prior to infusion, and then axi-cel (2 x 10 6 CART cells / kg). 9 Bridge therapy prior to lymphodepletion was at the investigator’s discretion. Disease response assessments (details previously described) were performed by the investigator, and at specified time points prior to the 24-month follow-up analysis, reviewed by an independent imaging review committee according to Lugano classification (details previously described), and after 24 months by the investigator only. All adverse events (AEs) were monitored through month 3 post-infusion, and thereafter only AEs of special interest (neurologic, hematologic, infectious, and autoimmune-related) were monitored through month 24; new and secondary malignancies were monitored through 15 years.

[0513] The primary endpoint for ZUMA-5 was ORR. Secondary endpoints included CR, duration of response (DOR), progression-free survival (PFS), overall survival (OS), time to next treatment (TTNT), safety, and CAR T cell blood levels. Exploratory endpoints included in this analysis were lymphoma-specific PFS and survival, where investigator-assessed progression events or death related to lymphoma, axi-cel, or lymphodepleting chemotherapy were considered events of interest. Clinical and pharmacokinetic outcomes were also evaluated in key patients and clinical subgroups, including pre-leukapheresis prior bendamustine use and baseline MTV.

[0514] Table 33: 3-year analysis results: DOR, PFS, and OS of follicular lymphoma patients by time point of bendamustine use prior to leukapheresis

[0515] The 3-year analysis for ZUMA-5 was conducted at a median follow-up of >36 months in enrolled FL patients. Efficacy outcomes were evaluated in all enrolled iNHL patients (intent-to-treat population); safety and transformation studies were evaluated in treated iNHL patients (laboratory and biomarker assessments were previously described). FL patients who received >3 lines of therapy (baseline central assessment excluded patients with other histology types) were evaluated in separate analyses. Patients who received retreatment with axi-cel were also evaluated separately (retreatment criteria were previously reported).

[0516] Baseline characteristics, responses, and incidence of AEs were summarized using descriptive statistics. Two-sided 95% CIs for response rates were estimated using the Clopper-Pearson method. Secondary endpoints involving time-to-event outcomes were estimated using the Kaplan-Meier method. Lymphoma-specific PFS and survival were estimated using the competing risks method, with events of interest considered primary events and deaths unrelated to lymphoma, axi-cel, or lymphodepleting chemotherapy considered competing risks. Event rates over time for primary events and competing risks were calculated by the cumulative incidence function.

[0517] Outcomes for FL patients were assessed by propensity score matching (PSM) by prior bendamustine use, accounting for distribution of baseline MTV, ECOG score, FLIPI score, number of prior chemotherapies, age, double refractory status, and whether the last systemic therapy was performed <12 months prior to leukapheresis. Association between CAR T cell levels and clinical outcomes was assessed using Wilcoxon rank-sum test.

[0518] Table 34: Clinical and transformational outcomes of follicular lymphoma patients by prior bendamustine use after propensity score matching

[0519] Table 35: PFS of FL patients by baseline metabolic tumor volume quartile

[0520] A total of 159 patients (127 FL, 31 MZL, 1 DLBCL) were enrolled and received leukapheresis, including 6 additional MZL patients enrolled after the 18-month analysis data cutoff date. Axi-cel was successfully manufactured for all enrolled patients. In addition to the previously described treatment-naive patients, 1 patient had disease transformation and 1 patient had no measurable disease. The patient with confirmed DLBCL did not receive axi-cel and was withdrawn from the study. As of the data cutoff date of March 31, 2022, a total of 152 patients received conditioning chemotherapy and axi-cel infusion (124 FL, 28 MZL).

[0521] Baseline characteristics for all 159 enrolled patients are not shown. The median age of FL patients was 60 years (range: 34-79 years) and the median age of MZL patients was 64 years (range: 43-77 years). Among FL patients, 56% were POD24 and 69% had prior exposure to bendamustine. Baseline characteristics for FL patients by prior bendamustine exposure (pre- and post-PSM) are shown in Table 31.

[0522] Table 36: 3-year efficacy outcome analysis by metabolic tumor volume study median and historical threshold Table 37: 3-year efficacy outcome analysis of follicular lymphoma patients by metabolic tumor volume quartile

[0523]

[0524]

[0525] *Matched using propensity score matching (1 : 1 matching) with log2 MTV caliper value = 1, age caliper value = 1.5, and exact match for PTR12MFL.†One observation for number of prior tre...

Claims

1. A method of predicting the likelihood of a patient in need thereof responding to a cell therapy product, comprising the steps of: quantitatively detecting the gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; and determining the likelihood of the patient responding to the cell therapy product based at least in part on the gene expression level, wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of response or a decreased likelihood of response compared to a predetermined likelihood of response rate, and wherein the gene expression level is quantitatively detected from a patient sample, and the patient sample is collected from the patient prior to treatment with the cell therapy product.

2. The method of claim 1, wherein the at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of response compared to a predetermined likelihood of response rate.

3. The method of claim 1, wherein the at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates an increased likelihood of response compared to a predetermined likelihood of response rate.

4. The method of claim 3, wherein the at least one gene exhibits an at least 20% elevated CD19 expression level relative to a CD19 control expression level, an at least 40% elevated MS4A1 expression level relative to a MS4A1 control expression level, and an at least 60% elevated TNFRSF17 expression level relative to a TNFRSF17 control expression level, indicating an increased likelihood of response compared to a predetermined likelihood of response rate.

5. The method of claim 1, wherein the at least one gene is selected from the group consisting of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2, and wherein an elevated gene expression level of the at least one gene compared to a control value indicates a decreased likelihood of response compared to a predetermined likelihood of response rate.

6. The method of any one of claims 1 to 5, wherein response is defined as one or more of complete response, partial response, durable response, progression free survival, or event free survival.

7. The method of any one of claims 1 to 6, wherein the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

8. The method of claim 7, wherein the cell therapy product is autologous or allogeneic.

9. The method of claim 7, wherein the target antigen is a tumor antigen, preferably selected from tumor-associated surface antigens such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD79a, CD79b, CD123, FLT3, BCMA, SLAMF7, CD8, CLL-1, c-Met, CMV-specific antigens, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal-epithelial mucin, EBV-specific antigens, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11 Ra, IL-13R-a2, influenza virus-specific antigens;CD38, insulin growth factor (IGF1)-l, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage- or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate-specific membrane antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stroma antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), GPC3 (glypican 3), and any derivative or variant of these antigens.

10. The method of claim 7, wherein the cell therapy product expresses a chimeric antigen receptor comprising a CD28 costimulatory domain.

11. The method of any one of claims 1 to 10, wherein the patient has been diagnosed with a cancer / tumor selected from the group consisting of a solid tumor, a sarcoma, a carcinoma, a lymphoma, a multiple myeloma, Hodgkin’s disease, non-Hodgkin’s lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (not specified type), follicular lymphoma (FL), DLBCL arising from FL, transformed follicular lymphoma, high grade B-cell lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), one or more of acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom’s macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, hepatocellular cancer, prostate cancer, breast cancer, or a combination thereof.

12. The method of claim 11, wherein the cancer is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

13. The method of any one of claims 1 to 12, wherein the cell therapy product is selected from the group consisting of axicinumab, belantumab, tisagenlecleucel, lisoceptumab, and bb2121.

14. The method of any one of claims 1 to 13, wherein the cell therapy product is administered as a second line therapy.

15. The method of any one of claims 1 to 14, wherein the patient sample is a tumor biopsy.

16. A method for treating a malignant tumor in a patient, comprising the steps of: quantitatively detecting a gene expression level of at least one gene selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, TCL1A, BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2; determining, at least partially based on the quantitatively detecting a gene expression level of at least one gene, whether an effective dose of a cell therapy product should be administered to the patient as a second line therapy or an effective dose of a cell therapy product should be administered to the patient as a third line therapy; and administering the effective dose of the cell therapy product as a second line therapy or as a third line therapy based on the determining step, wherein the gene expression level is quantitatively detected from a patient sample, and the patient sample is collected from the patient prior to treatment with the cell therapy product, wherein the patient is administered the effective dose of the cell therapy product as a second line therapy if the gene expression level of the at least one gene of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is at or above a control value of the at least one gene, or wherein the patient is administered the effective dose of the cell therapy product as a third line therapy if the gene expression level of the at least one gene of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is at or below a control value of the at least one gene, or wherein if the gene expression level of the at least one gene of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or below a control value for the at least one gene, the patient is administered the effective dose of the cell therapy product as a second line therapy, or wherein if the gene expression level of the at least one gene of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or above a control value for the at least one gene, the patient is administered the effective dose of the cell therapy product as a third line therapy.

17. The method of claim 16, wherein if the gene expression level of the at least one gene of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is below a control value for the at least one gene, or if the gene expression level of the at least one gene of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or above a control value for the at least one gene, the patient is administered a second line treatment regimen for a malignancy that does not include a cell therapy.

18. The method of claim 16, wherein the at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A.

19. The method of claim 18, wherein the at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17.

20. The method of any one of claims 16 to 19, wherein the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

21. The method of claim 20, wherein the cell therapy product is autologous or allogeneic.

22. The method of claim 20, wherein the target antigen is a tumor antigen, preferably selected from tumor-associated surface antigens such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD79a, CD79b, CD123, FLT3, BCMA, SLAMF7, CD8, CLL-1, c-Met, CMV-specific antigens, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal-epithelial mucin, EBV-specific antigens, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight-melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11 Ra, IL-13R-a2, influenza virus-specific antigens;CD38, insulin growth factor (IGF1)-l, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage- or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate-specific membrane antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stroma antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), GPC3 (glypican 3), and any derivative or variant of these antigens.

23. The method of claim 20, wherein the cell therapy product expresses a chimeric antigen receptor that comprises a CD28 costimulatory domain.

24. The method of any one of claims 16-23, wherein the patient has been diagnosed with a cancer / tumor selected from the group consisting of a solid tumor, a sarcoma, a carcinoma, a lymphoma, a multiple myeloma, Hodgkin’s disease, non-Hodgkin’s lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (not specified type), follicular lymphoma (FL), DLBCL arising from FL, transformed follicular lymphoma, high grade B-cell lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), one or more of acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom’s macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), monoclonal gammopathy of unknown significance (MGUS), plasmacytomas (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and Oki disease), head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, hepatocellular cancer, prostate cancer, breast cancer, or a combination thereof.

25. The method of claim 24, wherein the cancer is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) not specified type, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

26. The method of any one of claims 16-25, wherein the cell therapy product is selected from axicinumab, belantamab, tisotumab, lisoceptumab, and bb2121.

27. The method of any one of claims 16-26, wherein the patient sample is a tumor biopsy.

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