The influence of the tumor microenvironment on the effectiveness of immunotherapy

Abstract

This disclosure relates to the prognosis and influence of the tumor microenvironment on the efficacy of cell-based immunotherapies (e.g., T-cell, TCR-based, and CAR-based therapies).

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 490,870 filed on 17 March 2023; U.S. Provisional Patent Application No. 63 / 491,516 filed on 21 March 2023; U.S. Provisional Patent Application No. 63 / 496,887 filed on 18 April 2023; and U.S. Provisional Patent Application No. 63 / 502,295 filed on 15 May 2023 (each of which is incorporated herein by reference in its entirety).

[0002] (Field of invention) This disclosure relates to immunotherapy, compositions for immunotherapy, and methods for diagnosing and the prognosis of subjects receiving immunotherapy using the same. [Background technology]

[0003] Axi-cel is the first autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed / refractory (R / R) large B-cell lymphoma (LBCL) in adults after two or more systemic therapies. ZUMA-7 (NCT03391466) was the first randomized, global, multicenter phase 3 trial of axi-cel versus historical standard of care (SOC) as a second-line treatment in patients with R / R LBCL, where SOC consisted of two or three cycles of protocol-defined, investigator-selection platinum-based chemotherapy followed by high-dose chemotherapy with autologous stem cell transplantation (HDT-ASCT) for chemotherapy-sensitive patients. Axi-cel was superior to SOC, with a significantly improved efficacy and manageable safety profile. In the primary analysis, the event-free survival (EFS) hazard ratio (HR) was 0.398 (P<.0001; median EFS of 8.3 vs. 2.0 months and estimated 24-month EFS rates of 40.5% vs. 16.3% in the axi-cel vs. SOC arm, respectively). Despite these surprising results, a significant number of patients exhibited primary resistance (no response) or secondary resistance (relapse after initial response) to CAR-T cell therapy, necessitating further investigation of potential biomarkers associated with treatment resistance.

[0004] In LBCL, known prognostic factors in the clinical and real-world evidence of the chemoimmunotherapy era include high systemic tumor tissue volume, elevated lactate dehydrogenase (LDH), activated B cell (ABC)-like molecular subgroups, age, and systemic inflammatory markers such as interleukin-6 (IL-6) and C-reactive protein (CRP). In the cell therapy era, systemic tumor tissue volume and LDH were negatively associated with efficacy to CAR-T cell therapy, as demonstrated in ZUMA-1 (third-line or higher LBCL). Furthermore, the quality and quantity of pre-treatment tumor infiltration by T cells were positively associated with outcomes of CAR T cell therapy, as characterized by ImmunoSign 21 (IS21; T cell gene expression signature) and Immunoscore (immunohistochemistry [IHC] using CD3 and CD8 cells). Translational data from patients treated with CAR T-cell therapy in the real world further highlight the influence of tumor-associated chronic inflammation, checkpoint ligand upregulation, myelosuppression of CAR T-cell function, and the association between patterns of tumor genomic complexity and CAR T-cell outcomes. Nevertheless, predictive biomarkers for CAR T-cell intervention across treatment lines are not well established, and the association between tumor gene expression profiles and responses to CAR T-cells has not been comprehensively investigated. Furthermore, while the predictive and prognostic roles of TME are well described for solid tumors, the importance of the intratumoral immune status for CAR-T-cell therapy remains unclear.

[0005] To address these needs, we analyzed pre-treated tumor characteristics in ZUMA-7 to discover tumor-specific features that predict the effectiveness of axi-cel or SOC. [Overview of the Initiative]

[0006] It should be understood that the uses of this disclosure are not limited to the details described in the following embodiments, claims, description, and drawings. Other embodiments of this disclosure are possible, and it can be implemented or performed in numerous other ways.

[0007] This specification provides methods for evaluating the expression of specific biomarkers or analytes that may correlate with specific parameters after administration of immunotherapy (e.g., cell therapy), such as therapeutic outcomes including complete response (CR) or partial response (PR), or safety outcomes (e.g., adverse events) such as toxicity, neurotoxicity, or the occurrence of CRS. Methods are also provided for evaluating the likelihood of response and / or the likelihood of toxicity risk based on parameters, such as the expression of biomarkers or analytes in a patient. Furthermore, immunotherapies (e.g., T-cells, non-T-cells, TCR-based therapies, CAR-based therapies, bispecific T-cell engagers (BiTEs), and / or immune checkpoint blockers) including methods and uses of cells (e.g., engineered T-cells) and / or compositions thereof for the treatment of subjects having diseases or conditions that are generally cancerous or tumorous, or including them, such as leukemia or lymphoma. In some embodiments, the method and its use provide or achieve improved response and / or more sustained response or efficacy and / or reduced risk of toxicity or other side effects in subjects treated by several methods compared to a particular alternative method. In some embodiments, the method includes the administration of a specific number or relative number of manipulated cells, the administration of a specific type of cell in a predetermined ratio, the treatment of a specific patient population such as a patient population with a specific risk profile, stage classification, and / or prior treatment history, the administration of additional therapeutic agents, and / or combinations thereof.

[0008] In one aspect, this disclosure relates to an immunotherapy product. In a non-limiting example, one aspect of this disclosure relates to Yescarta as a second-line therapy. In one aspect, without being bound by any particular theory, the Phase 3 ZUMA-7 trial's overall survival (OS) analysis results, in which Yescarta demonstrated a statistically significant improvement in OS compared to historical treatment, represented nearly 30 years of standard care (SOC) in a treatment setting for second-line relapsed / refractory large B-cell lymphoma (R / R LBCL) within 12 months of completion of first-line therapy. This is a multi-step process including a platinum-based salvage combination chemoimmunotherapy regimen followed by high-dose therapy (HDT) and stem cell transplantation (ASCT) in patients responding to salvage chemotherapy. OS was designated as a clinically important pre-identified critical secondary endpoint, defined as the length of time from randomization to death from any cause.

[0009] In one embodiment, the present disclosure provides a method for predicting the likelihood of response to a cell therapy product in a patient in need thereof, comprising: quantifying 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, at least partially, the likelihood of response to the cell therapy product in the patient from the gene expression level. In such embodiments, an increase in the gene expression level of at least one gene compared to a control value indicates an increase or decrease in the likelihood of response compared to a predetermined likelihood of response rate, the gene expression level is quantified from a patient sample, the patient sample is collected from the patient before treatment with the cell therapy product.

[0010] In one embodiment, the present disclosure provides a method for treating a patient's malignant tumor, comprising: quantifying 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; determining, at least partially, whether to administer an effective dose of the cell therapy product to the patient as a second-line therapy or as a third-line therapy based on the quantification of the gene expression level of at least one gene; and administering an effective dose of the cell therapy product as a second-line therapy or as a third-line therapy based on the determination step. In certain embodiments, if the expression level of at least one of the following genes is greater than or equal to the control value for at least one gene, the patient is administered an effective dose of the cell therapy product as a second-line therapy, or if the expression level of at least one of the following genes is less than or equal to the control value for at least one gene, the patient is administered an effective dose of the cell therapy product as a third-line therapy, or BNIP3L If the expression levels of MXI1, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 are below the control value for at least one gene, or if the expression level of at least one of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is above the control value for at least one gene, the patient will be administered an effective dose of the cell therapy product as third-line therapy.

[0011] In one embodiment, the immunotherapy is T-cell therapy. In some embodiments, the T-cell therapy includes adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, modified autologous cell therapy (eACT), and allogeneic T-cell transplantation. In a particular embodiment, eACT includes the administration of modified antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, eACT includes the administration of modified antigen-specific T cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is CAR T-cell or TCR T-cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T-cell therapy.

[0012] The following are non-limiting embodiments of this disclosure.

[0013] In at least one aspect, the present disclosure provides a method for predicting the likelihood of a response to a cell therapy product in a patient in need of it. 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 is quantified. To determine, at least partially, the potential response to cell therapy products in patients from gene expression levels, Includes, An increase in the gene expression level of at least one gene compared to the control value indicates the possibility of an increased or decreased response, compared to a predetermined probability of response rate. Gene expression levels are quantified from patient samples, and these samples are collected from patients before treatment with cell therapy products.

[0014] In one embodiment, at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A, and an increase in the gene expression level of at least one gene compared to a control value indicates an increase in the likelihood of response compared to a predetermined likelihood of response rate.

[0015] In one embodiment, at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17, and an increase in the gene expression level of at least one gene compared to a control value indicates an increase in the likelihood of response compared to a predetermined likelihood of response rate.

[0016] In one embodiment, at least one gene exhibits an increase of at least 20% in CD19 expression level compared to the control expression level of CD19, an increase of at least 40% in MS4A1 expression level compared to the control expression level of MS4A1, and an increase of at least 60% in TNFRSF17 expression level compared to the control expression level of TNFRSF17, indicating an increased likelihood of response compared to a predetermined likelihood of response rate.

[0017] In one embodiment, 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 an increase in the gene expression level of at least one gene compared to a control value indicates a decrease in the likelihood of response compared to a predetermined likelihood of response rate.

[0018] In some embodiments, a response is defined as one or more of the following: complete response, partial response, ongoing response, progression-free survival, or event-free survival.

[0019] In one embodiment, the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

[0020] In some embodiments, the cell therapy product is autologous or allogeneic.

[0021] In one embodiment, the target antigen is preferably a tumor-associated surface antigen, such as 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, β-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 antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, or EGFR variant. EGFRvIII, ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-related protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-related antigen, sphingoglycolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-related antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGF-II receptor, IGF-II, IL-11Rα, IL-13Rα2, influenza virus-specific antigen;CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, κ chain, LAGA-1a, λ chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigen, e.g., 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, variant hsp70-2, variant p53, variant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 The tumor antigens are selected from antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivorin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gp120), GPC3 (glypican 3), and any derivatives or variants of these antigens.

[0022] In one embodiment, the cell therapy product expresses a chimeric antigen receptor containing a CD28 costimulatory domain.

[0023] In some cases, the patient may have solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (non-specific 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 bone Myelin 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"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular Lymphoma, pilocytic cell leukemia, small cell or large cell follicular lymphoma, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal dysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, plasmacytoproliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance Diagnosed with cancer / tumor selected from the group consisting of gynoplasmosis (MGUS), plasmacytoma (e.g., plasmacytoplasmic hyperplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), 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 carcinoma, prostate cancer, breast cancer, or combinations thereof.

[0024] In some cases, the cancer is (recurrent or refractory) diffuse large B-cell lymphoma (DLBCL) nonspecific type, mediastinal large B-cell lymphoma, high-grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

[0025] In one embodiment, the cell therapy product is selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagenlecleucel, lysocabbutagen maralucel, and bb2121.

[0026] In some cases, cell therapy products are administered as a second-line therapy.

[0027] In one embodiment, the patient sample is a tumor biopsy.

[0028] In some aspects, this disclosure provides a method for treating a patient's malignant tumor. 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 is quantified. At least partially, by quantifying the gene expression level of at least one gene, it is possible to determine whether an effective dose of cell therapy product should be administered to a patient as a second-line therapy or as a third-line therapy, Based on the determination process, the effective dose of the cell therapy product is administered as a second-line or third-line therapy. Includes, Gene expression levels are quantified from patient samples, and patient samples are collected from patients before treatment with cell therapy products. If the expression level of at least one of the following genes—CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A—is equal to or greater than the control value for at least one gene, the patient will be administered an effective dose of a cell therapy product as a second-line therapy, or If the expression level of at least one of the following genes—CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A—is below the control value for at least one gene, the patient will be administered an effective dose of a cell therapy product as a third-line therapy, or If the expression level of at least one of the following genes is below the control value for at least one gene: BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2, the patient will be administered an effective dose of the cell therapy product as a second-line therapy, or If the expression level of at least one of the following genes is greater than or equal to the control value for at least one gene, the patient will be administered an effective dose of the cell therapy product as a third-line therapy.

[0029] In one embodiment, if the expression level of at least one gene among CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is below the control value for at least one gene, or if the expression level of at least one gene among BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 is at or above the control value for at least one gene, the patient is administered a second-line course of therapy for malignant tumors that does not include cell therapy.

[0030] In one embodiment, at least one gene is selected from the group consisting of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A.

[0031] In one embodiment, at least one gene is selected from the group consisting of CD19, MS4A1, and TNFRSF17.

[0032] In one embodiment, the cell therapy product is a CAR T or TCR T cell therapy that recognizes a target antigen.

[0033] In some embodiments, the cell therapy product is autologous or allogeneic.

[0034] In one embodiment, the target antigen is preferably a tumor-associated surface antigen, such as 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, β-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 antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, or EGFR variant. EGFRvIII, ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-related protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-related antigen, sphingoglycolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-related antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGF-II receptor, IGF-II, IL-11Rα, IL-13Rα2, influenza virus-specific antigen;CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, κ chain, LAGA-1a, λ chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigen, e.g., 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, variant hsp70-2, variant p53, variant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 The tumor antigens are selected from antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivorin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gp120), GPC3 (glypican 3), and any derivatives or variants of these antigens.

[0035] In one embodiment, the cell therapy product expresses a chimeric antigen receptor containing a CD28 costimulatory domain.

[0036] In some cases, the patient may have solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (non-specific 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 bone Myelin 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"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular Lymphoma, pilocytic cell leukemia, small cell or large cell follicular lymphoma, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal dysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, plasmacytoproliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance Diagnosed with cancer / tumor selected from the group consisting of gynoplasmosis (MGUS), plasmacytoma (e.g., plasmacytoplasmic hyperplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), 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 carcinoma, prostate cancer, breast cancer, or combinations thereof.

[0037] In some cases, the cancer is (recurrent or refractory) diffuse large B-cell lymphoma (DLBCL) nonspecific type, mediastinal large B-cell lymphoma, high-grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

[0038] In one embodiment, the cell therapy product is selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagenlecleucel, lysocabbutagen maralucel, and bb2121.

[0039] In one embodiment, the patient sample is a tumor biopsy.

[0040] In one embodiment, a method for treating a subject having lymphoma is disclosed, which includes administering a dose-reducing regimen to the subject and administering immunotherapy to the subject in accordance with the dose-reducing regimen.

[0041] In one embodiment, the weight loss regimen includes one of the options included in Table 39. In another embodiment, the weight loss regimen includes at least two of the options included in Table 39.

[0042] In some embodiments, immunotherapy includes anti-CD19 CAR T cells.

[0043] In one embodiment, if the subject has a systemic tumor tissue volume exceeding a predetermined level, the subject is administered a dose-reducing regimen. In another embodiment, if the subject has a systemic tumor tissue volume below a predetermined level, the subject is not administered a dose-reducing regimen.

[0044] In one embodiment, the disclosure relates to a method for predicting the likelihood of response to a cell therapy product in a patient in need thereof, comprising: quantifying the gene expression levels 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 includes adding the gene expression levels of the at least two genes; and determining, at least in part, the likelihood of response to the cell therapy product in the patient from the composite expression score. In such embodiments, an increase in the composite expression score compared to a control value indicates an increase in the likelihood of response compared to a predetermined likelihood of response rate. In such embodiments, the composite expression score is calculated as follows: the individual expression levels of 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 of the at least two genes. Next, the generated composite expression score is compared to a control value, where the control value is the historical median of past composite expression scores for at least two genes from other patients.

[0045] In one embodiment, the disclosure relates to a method for predicting the likelihood of response to a cell therapy product in a patient for whom it is necessary to predict the likelihood of response to a cell therapy product, and includes: quantifying the gene expression levels 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 includes adding the gene expression levels of at least two genes; and determining, at least in part, the likelihood of response to the cell therapy product in the patient from the composite expression score. In such embodiments, an increase in the composite expression score compared to a control value indicates a decrease in the likelihood of response compared to a predetermined likelihood of response rate. In such embodiments, the composite expression score is calculated as follows: The individual expression levels of at least two genes derived 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 at least two genes. The generated composite expression score is then compared to a control value, where the control value is the historical median of past composite expression scores for at least two genes from other patients.

[0046] In one embodiment, the present disclosure relates to a method for treating a patient's malignant tumor, comprising: quantifying the gene expression levels 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 includes adding the gene expression levels of at least two genes; determining, at least partially, from the composite expression score whether the patient should be administered an effective dose of the cell therapy product as a second-line therapy or as a third-line therapy; and administering an effective dose of the cell therapy product as a second-line therapy or as a third-line therapy based on the determination step. In such embodiments, if the composite expression score of at least two of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is greater than or equal to the control value, the patient is administered an effective dose of the cell therapy product as second-line therapy, or if the composite expression score of at least two of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is less than or equal to the control value, the patient is administered an effective dose of the cell therapy product as third-line therapy. In such embodiments, the composite expression score is calculated as follows: The individual expression levels of 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 at least two genes. Next, the generated composite expression score is compared to a control value, where the control value is the historical median of past composite expression scores for at least two genes from other patients.

[0047] In one embodiment, the disclosure relates to a method for treating a patient's malignant tumor, and includes: quantifying the gene expression levels 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 includes adding the gene expression levels of at least two genes; determining, at least partially, from the composite expression score whether the patient should be administered an effective dose of the cell therapy product as a second-line therapy or as a third-line therapy; and administering an effective dose of the cell therapy product as a second-line therapy or as a third-line therapy based on the determination step. In such embodiments, 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 is below the control value, the patient is administered an effective dose of the cell therapy product as second-line therapy. 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 is above the control value, the patient is administered an effective dose of the cell therapy product as third-line therapy. In such embodiments, the composite expression score is calculated as follows. The individual expression levels of at least two genes derived 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 at least two genes. The generated composite expression score is then compared to a control value, where the control value is the historical median of past composite expression scores for at least two genes from other patients. [Modes for carrying out the invention]

[0048] This disclosure is based in part on the finding that the pre-therapy gene expression levels of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A are positively associated with an increased likelihood of response to cell therapy, and that the pre-therapy gene expression levels of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 are inversely correlated with the likelihood of response to cell therapy. The findings of this invention inform whether a patient should be administered cell therapy.

[0049] In one aspect, this disclosure relates to an immunotherapy product. In a non-limiting example, one aspect of this disclosure relates to Yescarta as a second-line therapy. In one aspect, without being bound by any particular theory, the Phase 3 ZUMA-7 trial's overall survival (OS) analysis results, in which Yescarta demonstrated a statistically significant improvement in OS compared to historical treatment, represented nearly 30 years of standard care (SOC) in a treatment setting for second-line relapsed / refractory large B-cell lymphoma (R / R LBCL) within 12 months of completion of first-line therapy. This is a multi-step process including a platinum-based salvage combination chemoimmunotherapy regimen followed by high-dose therapy (HDT) and stem cell transplantation (ASCT) in patients responding to salvage chemotherapy. OS was designated as a clinically important pre-identified critical secondary endpoint, defined as the length of time from randomization to death from any cause.

[0050] definition To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of these terms and other terms are provided throughout this specification.

[0051] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise.

[0052] Where used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.”

[0053] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two designated features or components, with or without the other. Accordingly, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0054] As used herein, the terms “for example” and “that is” are used merely as examples and are not intended to be limiting, and should not be construed as referring only to items explicitly listed herein.

[0055] Terms like "greater than or equal to," "at least," and "greater than," for example, "at least one," are not limiting, but rather mean at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 3 8, 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, 9 1, 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, 1 It is understood to include 33, 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 any value greater than the listed value. Any larger number or fraction in between is also included.

[0056] Conversely, the term "less than or equal to" includes each value smaller than the listed value. For example, "100 or fewer 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, 5 This includes 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any fewer number or fraction in between is also included.

[0057] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limiting, but they mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9 0, 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 It is understood to include 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.

[0058] Throughout this specification, the word “comprising” or variations such as “comprises” or “comprising” is understood to mean including the elements, integers, or processes, or groups of elements, integers, or processes described herein, but not to mean excluding any other elements, integers, or processes, or groups of elements, integers, or processes. Whenever an aspect is described herein using the word “comprising,” it is understood that other similar aspects described using the terms “consisting of” and / or “consisting essentially of” are also presented. The term “consisting of” excludes any elements, processes, or components not specified in the claims. In re Gray, 53 F.2d 520,11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448,450 (Bd.App.1948) ("consisting of" is defined as "closing the claim to the inclusion of materials other than those described, excluding impurities that are usually associated with them"). The term "essentially consisting of" limits the scope of the claim to the materials or processes described and "not substantially affecting the basic and novel features(s) of the claimed disclosure."

[0059] As used herein, unless otherwise specified or evident from the context, the term “about” means a value or composition that is within the acceptable margin of error of a particular value or composition as determined by those skilled in the art, and this depends to some extent on how such value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “about” or “approximately” may mean within one or two standard deviations or more, according to convention in the art. “About” or “approximately” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may encompass any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, this term may mean up to an order of magnitude off or up to five times the value. Where specific values ​​or compositions are presented in this disclosure, unless otherwise specified, the meaning of “approximately” or “about” should be assumed to be within the acceptable margin of error for those specific values ​​or compositions.

[0060] As described herein, any range of concentration, percentage, ratio, or integer should be understood to include any integer values ​​within the listed ranges, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0061] The units, prefixes, and symbols used herein are presented in the format accepted by the Systeme International de Unites (SI). Numerical ranges include the number that defines the range.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to whom this disclosure relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology," 5th ed., (2013), Academic Press; and "The Oxford Dictionary of Biochemistry and Molecular Biology," Cammack et al. eds., 2nd ed., (2006), Oxford University Press, provide those skilled in the art with many of the terms used herein.

[0063] "Administer" refers to the physical delivery of a drug to a target using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration by injection or infusion. Exemplary routes of administration for the compositions disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration by injection or infusion. As used herein, the term "parenteral administration" generally means, but is not limited to, methods of administration other than enteral and topical administration by injection, including, intravenous, intramuscular, intra-arterial, subarachnoid, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-intravenous route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods. In one embodiment, CAR T cell therapy is administered by an "infusion product" containing CAR T cells.

[0064] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains linked together 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 light 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 may be further subdivided into hypervariable regions called "complementarity-determining regions" (CDRs), with more conserved regions called "framework regions" (FRs) interposed between them. Each VH and VL contains three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0065] Antibodies include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, modified antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing 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, intracellular antibodies, antibody fusions (sometimes referred to as "antibody conjugates" in this specification), heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies, or single-chain Fv (single-chain) antibodies. Examples include Fv, scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-bound Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetic”), and any of the above antigen-binding fragments. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies.

[0066] The terms “antigen-binding molecule,” “antigen-binding portion,” or “antibody fragment” refer to any molecule containing the antigen-binding portion (e.g., CDR) of an antibody from which the molecule is derived. An antigen-binding molecule may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to CD19. In further embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen and contains one or more of its complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule includes or consists of an avimer.

[0067] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may involve either antibody production or activation of specific immune cells, or both. Those skilled in the art will readily understand that virtually all proteins or peptides, and any macromolecule, can function as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or by recombination. Antigens may be specific to certain tissues, such as cancer cells, or they may be expressed broadly. Furthermore, larger molecular fragments can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0068] The term "neutralize" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the ligand's biological action. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or otherwise alters the ligand's ability to bind by indirect means (such as structural or energy changes within the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0069] The term “autologous” refers to any material derived from the same individual that is later reintroduced. For example, the method of manipulated autologous cell therapy (eACT®) described herein involves harvesting lymphocytes from a patient, which are then manipulated to express, for example, a CAR construct, and then administered to the same patient.

[0070] The term "allogeneic" refers to any material that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

[0071] In one embodiment, CAR T cell therapy includes "axicabutagen silol-ucel therapy." "Axicabutagen silol-ucel therapy" is 2 × 10 6 The treatment consists of a single infusion of autologous T cells transduced to anti-CD19 CAR, administered intravenously at a targeted dose of 100 x 10⁶ anti-CD19 CAR T cells / kg. For subjects weighing over 100 kg, the maximum fixed dose is 2 × 10⁶. 8Individual anti-CD19 CAR T cells may be administered. Anti-CD19 CAR T cells are autologous human T cells engineered to express an extracellular single-chain variable fragment (scFv) linked to a serially arranged intracellular signaling region consisting of signaling domains derived from CD28 and CD3ζ (CD3 zeta) molecules, and possessing specificity for CD19. The anti-CD19 CAR vector construct was designed, optimized, and first tested in the Department of Surgery at 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 is added because mouse models suggest it is important for the antitumor effect and persistence of anti-CD19 CAR T cells (Kowolik et al, Cancer Res. 2006;66(22):10995-1004). The signaling domain of the CD3ζ chain is used for T cell activation. These fragments were cloned into a mouse stem cell virus-based vector (MSGV1) and used to genetically engineer autologous T cells. The CAR construct is inserted into the T cell genome by retroviral vector transduction. In summary, peripheral blood mononuclear cells (PBMCs) are obtained by leukocyte apheresis and Ficol isolation. Peripheral blood mononuclear cells are activated by culturing with anti-CD3 antibody in the presence of recombinant interleukin 2 (IL-2). Stimulated cells are transduced with a retroviral vector containing the anti-CD19 CAR gene and grown in culture to produce T cells that are sufficiently engineered for administration. In some embodiments, CAR T cell therapy is Yescarta® (Axicapbutagensilolucel).In some aspects, CAR T-cell therapy is Tecartus® (brexkabutagen autolucell).

[0072] The terms “transduction” and “transduced” refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0073] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and proliferation can lead to the formation of malignant tumors, which may invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may encompass tumors. In this application, the term cancer is synonymous with malignant tumor. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein include, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, It may be used to reduce the tumor size of tumors resulting from penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, axial vertebral tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of the above cancers. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is NHL. Certain cancers may be responsive to chemotherapy or radiotherapy, or certain cancers may be resistant to treatment. Treatment-resistant cancer refers to cancer that is not suitable for surgical intervention, either because it is unresponsive to chemotherapy or radiotherapy from the outset, or because it becomes unresponsive over time.

[0074] As used herein, “antitumor effect” refers to a biological effect that may manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall survival or progression-free survival, an extension of life expectancy, or improvement of various physiological symptoms associated with tumor. The antitumor effect may also refer to the prevention of tumor development, for example, by a vaccine.

[0075] As used herein, “cytokine” refers to a non-antibody protein released by a 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” means a protein released by a population of cells that acts on another cell as an intercellular mediator. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma.Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0076] Chemokines are a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus and activation-regulated chemokines (TARC or CCL17).

[0077] As used herein, “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) containing a binding domain capable of interacting with a specific tumor antigen enable T cells to target and kill cancer cells expressing a specific tumor antigen. In one embodiment, T cell therapy is based on T cells engineered to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) comprising (i) an antigen-binding molecule, (ii) a costimulatory domain, and (iii) an activating domain. The costimulatory domain may include an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain may include a hinge domain that can be shortened.

[0078] The therapeutic dose, effective dose, effective amount, effective dose, or therapeutically effective dosage of a therapeutic agent, such as engineered CAR T cells, small molecules, or "agent" as described herein, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of the disease, promotes disease regression as demonstrated by a reduction in the severity of disease symptoms, increases the frequency and duration of asymptomatic periods of the disease, or prevents functional or physical impairment resulting from the distress of the disease. Such terms may be used interchangeably. The ability of a therapeutic agent to promote disease regression may be evaluated using various methods known to those skilled in the art, for example, in human subjects in clinical trials, in animal model systems to predict efficacy in humans, or by analyzing the activity of the agent in in vitro assays. The therapeutic dose and dosage regimen may be empirically determined by testing in known in vitro or in vivo (e.g., animal model) systems.

[0079] The term “combination” refers to either a fixed combination in a single unit dosage form, or a combination administration in which the compound of this disclosure and a co-administered agent (e.g., another drug, also referred to later, “therapeutic” or “drug”) may be administered independently and simultaneously, or separately within a time interval, where these time intervals in particular allow the co-administered agent to exhibit a synergistic effect. Individual components may be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) may be reconstituted or diluted to a desired dose before administration. As used herein, the terms “co-administration” or “combination administration,” etc., are intended to include administration of a selected co-administered agent to a single target (e.g., a patient) to which administration is required, and are intended to encompass treatment plans in which the drugs are not necessarily administered via the same route of administration or simultaneously.

[0080] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or whose benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and must not be harmful to the recipient or whose benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that is involved in the transport or delivery of a drug from one part of the body to another (for example, from one organ to another). Each carrier present in a pharmaceutical composition must be compatible with the other components of the formulation and "acceptable" in the sense that it is not harmful to the patient, or whose benefits to the recipient outweigh any harmful effects. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; 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 laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0081] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant subjects or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, but is not limited to, forms adapted for: oral administration, e.g., liquid (aqueous or non-aqueous or suspension), tablets, e.g., buccal, sublingual, and those targeting systemic absorption, bolus, powder, granules, paste for application to the tongue; parenteral administration, e.g., sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity; intravaginal or rectal, e.g., as a pessary, cream, or foam; sublingual; ocular; transdermal; or transnasal, lung, and other mucosal surfaces.

[0082] The terms “reduce” and “decrease” are used interchangeably herein and refer to any change that becomes less than the original. “Reduce” and “decrease” are relative terms and require a comparison between before and after measurement. “Reduce” and “decrease” include complete depletion.

[0083] The term “reference” describes the standard or control on which the comparison is performed. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control which is a drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a reference or control from the past that is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation, where the similarity is sufficient to justify the dependence on and / or comparison with the selected reference or control.

[0084] Throughout this text, the “control value” refers to the historical value of a particular analyte observed in a population prior to administration of the cell therapy product. In some embodiments, deviations from historical values ​​correlate with an increase or decrease in the likelihood of a response to the cell therapy product in a particular patient relative to a predetermined and / or historical likelihood of a response to the cell therapy product. More specifically, in some embodiments, an increase in the expression level of an analyte in a test sample from a patient versus the control expression level of its corresponding analyte is related to an increase in the likelihood of a response to the cell therapy product in that patient relative to a predetermined and / or historical likelihood of a response to the cell therapy product. In certain embodiments, the increased likelihood of a response is measured relative to the known historical mean likelihood of a response to the cell therapy product in the population. In some embodiments, an increase in the expression level of an analyte in a test sample from a patient versus the control expression level of its corresponding analyte is related to a decrease in the likelihood of a response to the cell therapy product in that patient relative to a predetermined and / or historical likelihood of a response to the cell therapy product. In certain embodiments, the decrease in the likelihood of a response is measured relative to the known and historical mean likelihood of a response to the cell therapy product in the population.

[0085] As used herein, the term “predetermined” refers to an expected or possible outcome based on information that does not include specific information about any particular patient who may or could be a candidate for cell therapy.

[0086] The terms “product” or “injection product” are used interchangeably herein and refer to a T cell composition administered to a target subject requiring administration. For example, in CAR T cell therapy, the T cell composition is administered as an injection product.

[0087] As used herein, the term “lymphocyte” encompasses natural killer (NK) cells, T cells, and B cells. NK cells are a type of cytotoxic lymphocyte that are the main component of the innate immune system. NK cells reject tumor and virus-infected cells. They act through the process of apoptosis or programmed cell death. They are called “natural killers” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (immunity without antibody involvement). Their T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a differentiated organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells, namely helper T cells (e.g., CD4+ cells), cytotoxic T cells (TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell, or also known as killer T cell), and memory T cells ((i) Stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and memory cells Cells exhibit numerous functional characteristics specific to each cell: (ii) Central memory TCM cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; however, (iii) Effector memory TEM cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies).

[0088] Furthermore, each type of T cell can be characterized using cell surface markers well known in the art. 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 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+. A further marker for effector memory T cells is IL-2Rβ+. For central memory T cells (Tcm), preferred markers include CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For effector T cells (Teff), preferred markers include, but are not limited to, CD45RA+, CD95+, IL-2Rβ+, CCR7-, and CD62L-.

[0089] The terms “genetically modified” or “modified” refer to methods of altering the genome of a cell, including but not limited to deleting coding regions or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In some embodiments, the modified cells are lymphocytes, such as T cells, which may be obtained from either a patient or a donor. The cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which are incorporated into the cell’s genome.

[0090] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including alpha, cytokines, and complement) produced by either these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of a vertebrate of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0091] The term "immunotherapy" refers to the treatment of a person who is suffering from a disease or at risk of developing or relapsing from a disease, by means of methods including inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. Examples of T-cell therapy include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT®), and allogeneic T-cell transplantation. However, those skilled in the art will recognize that the conditioning methods disclosed herein enhance the efficacy of any transplanted T-cell therapy. Examples of T-cell therapies are described in U.S. Patent Publications 2014 / 0154228 and 2002 / 0006409, U.S. Patents 7,741,465, 6,319,494, 5,728,388, International Publications 2008 / 081035, 2015 / 20096, 2016 / 191756, 2016 / 191755, 2019 / 079564, and 2021 / 092290, each of which is incorporated herein in its entirety. In some embodiments, the immunotherapy includes CAR T-cell therapy. In some embodiments, the CAR T-cell therapy product is administered via infusion.

[0092] T cells for immunotherapy may be derived from any source known in the art. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population, or T cells may be obtained from a subject. T cells may be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In addition, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from blood units taken from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. Further methods for isolating T cells for T cell therapy, and for producing CAR T cells for cell therapy, are disclosed in U.S. Patent Publication No. 2013 / 0287748, International Publication No. 2015 / 20096, International Publication No. 2016 / 191756, International Publication No. 2016 / 191755, International Publication No. 2019 / 079564, and International Publication No. 2021 / 092290, each of which is incorporated herein by reference in its entirety.

[0093] The term "modified autologous cell therapy" or "eACT®," also known as adoptive cell transfer, is a process in which a patient's own T cells are collected and then genetically modified to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells can be modified, for example, to express chimeric antigen receptors (CARs). CAR-positive (+) T cells are modified to express extracellular single-strand variable fragments (scFv) specific to a particular tumor antigen, linked to an intracellular signaling region containing at least one costimulatory domain and at least one activating domain. CAR scFv can be designed to target, for example, cells of the B cell lineage, including all normal B cells, and, but not limited to, B cell malignancies, including diffuse large B-cell lymphoma (DLBCL) nonspecific type, 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 Applications Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated in their entirety by reference.

[0094] As used herein, “patient” or “subject” encompasses any human being suffering from cancer (e.g., lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably herein.

[0095] As used herein, the term “in vitro cells” refers to any cells cultured ex vivo. In particular, in vitro cells may include T cells. The term “in vivo” means within a patient.

[0096] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, of which there are many types, generally called proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0097] As used herein, “stimulus” refers to a primary response induced by the binding of a stimulating molecule to its homologous ligand, where the binding mediates a signaling event. “Stimulating molecule” is a molecule on a T cell, such as a T cell receptor (TCR) / CD3 complex that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell. “Stimulating ligand” is a ligand that, when present on an antigen-presenting cell (e.g., APCs, dendritic cells, B cells), specifically binds to a stimulating molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, and proliferation. Examples of stimulating ligands include, but are not limited to, anti-CD3 antibodies, peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0098] As used herein, “co-stimulatory signal” refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, elicits an upregulation or downregulation of a T cell response, such as, but not limited to, proliferation and / or major molecules.

[0099] As used herein, “costimulatory ligand” encompasses molecules on antigen-presenting cells that specifically bind to homologous costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses, such as proliferation, activation, and differentiation, but is not limited to these. In addition to the primary signals provided by stimulatory molecules, costimulatory ligands induce signals, for example, by binding the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Co-stimulatory ligands may include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, 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), ligands that specifically bind to B7-H3, lymphotoxin β receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed cell death (PD)L1. In certain embodiments, the co-stimulatory ligand may include, but are not limited to, a co-stimulatory molecule present on T cells, such as ligands that specifically bind to, but are not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or an antibody that specifically binds to tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0100] "Co-stimulatory molecules" are congenital binding partners on T cells that specifically bind to costimulatory ligands and thereby mediate costimulatory responses by T cells, such as proliferation, but are not limited to these. Examples of 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, and CD276 (B7-H3). , CD28, CD29, CD3 (α; β, δ, ε, gamma, ζ), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, C D80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), M Examples include HC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleaved forms, or combinations thereof.

[0101] The terms “reduce” and “decrease” are used interchangeably herein and refer to any change that becomes less than the original. “Reduce” and “decrease” are relative terms and require a comparison between before and after measurement. “Reduce” and “decrease” include complete depletion. Similarly, the term “increase” refers to any change that becomes higher than the original value. “Increase,” “higher,” and “lower” are relative terms and require a comparison between before and after measurement and / or between reference standards. In some embodiments, the reference value is obtained from a general population, which may be the general population of patients. In some embodiments, the reference value is derived from an quartile analysis of the general patient population.

[0102] "Treatment" or "to treat" a subject means any type of intervention or process performed on the subject, or administration of an active agent to the subject, for the purpose of reducing, reducing, improving, inhibiting, delaying, or preventing the onset, progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with the disease. In some embodiments, "treatment" or "to treat" includes partial remission. In other embodiments, "treatment" or "to treat" includes complete remission. In some embodiments, treatment may be prevention, in which case the treatment is administered before any symptoms of the condition are observed. As used herein, the term "prevention" means prevention of disease or condition or protective treatment against them. Prevention of symptoms, disease, or condition may include, for example, a reduction (e.g., mitigation) of one or more symptoms of the disease or condition compared to a reference level (e.g., symptoms in a similar subject that is not treated). Prevention may include, for example, delaying the onset of one or more symptoms of the disease or condition compared to a reference level (e.g., the onset of symptoms in a similar untreated subject). In embodiments, the disease is a disease described herein. In some embodiments, the disease is cancer. In some embodiments, the pathological condition is CRS or neurotoxicity. In some embodiments, indicators of improvement or treatment success may include a judgment that the patient did not show a corresponding score on a toxicity rating scale (e.g., CRS or neurotoxicity rating scale), for example, a score of less than 3, or a change in grade or severity on the rating scale described herein, for example, 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.

[0103] As used herein, “myeloid cells” refers to a subgroup of leukocytes, including granulocytes, monocytes, macrophages, and dendritic cells.

[0104] In one embodiment, the terms “high” and “low” mean “above” and “below” the median of a representative population. In one embodiment, these terms mean the upper quartile or the lower quartile, respectively. Both the mean and the quartile distribution can be determined by those skilled in the art by commonplace methods.

[0105] As used herein, the term “quartile” is a statistical term that describes the division of a data set into four predefined intervals based on the difference between the data value and the entire set of those observations.

[0106] As used herein, the term "Day 0 of the study" is defined as the day on which a subject first receives CAR T cell infusion. The day before Day 0 of the study is Day 1 of the study. Any day after registration and prior to Day 1 of the study is continuous and a negative integer.

[0107] As used herein, the term “sustained response” refers to a subject who has maintained a response for at least one year of follow-up after CAR T cell injection. In one embodiment, “response period” is defined as the time from the first objective response to death due to disease progression or disease recurrence.

[0108] As used herein, the term “relapse” refers to a patient who has achieved a complete response (CR) or a partial response (PR) and subsequently experienced disease progression.

[0109] As used herein, the term “ineffective” refers to subjects who have not experienced a complete response (CR) or partial response (PR) after CAR T-cell infusion, including subjects with stable disease (SD) and disease progression (PD).

[0110] As used herein, the term “objective response” refers to complete response (CR), partial response (PR), or no response. This may be evaluated according to the revised IWG Response Criteria for Malignant Lymphoma (Cheson et al., J Clin Oncol. 2007;25(5):579-86).

[0111] As used herein, the term “complete response” refers to the complete recovery of the disease, which becomes undetectable by radioimaging and clinical laboratory evaluation. There are no signs of cancer at a given time.

[0112] As used herein, the term “partial response” refers to a reduction of more than 30% of the tumor without complete recovery.

[0113] As used herein, the "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).

[0114] As used herein, “progression-free survival (PFS)” may 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 investigator’s assessment of response as defined by the IWG criteria (Cheson et al., J Clin Oncol. 2007;25(5):579-86).

[0115] The term "overall survival (OS)" can be defined as the time from the date of T cell infusion to the date of death due to any cause.

[0116] When used herein, the proliferation and persistence of peripheral blood CAR T cells can be monitored by qPCR analysis using CAR-specific primers targeting the scFv portion of the CAR (e.g., the heavy chain of the CD19-binding domain) and its hinge / CD28 transmembrane domain. Alternatively, this can be measured by counting the CAR cell / blood unit volume.

[0117] As used herein, the schedule for blood collection for CAR T cells may be before CAR T cell infusion, on 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).

[0118] As used herein, “CAR T cell peak” is defined as the maximum absolute number of CAR+PBMCs / μL in serum achieved after day 0.

[0119] As used herein, “CAR T cell peak time” is defined as the number of days from day 0 to the day on which the CAR T cell peak is achieved.

[0120] As used herein, "Area Under Curve (AUC) of CAR T cell levels from day 0 to day 28" is defined as the area under the curve on a plot of CAR T cell levels against scheduled visits from day 0 to day 28. This AUC represents the total level of CAR T cells over a long period of time.

[0121] When used herein, the schedule for blood collection for cytokines is the day before or the day of conditioning chemotherapy (days 1-5), day 0, day 1, day 3, day 5, day 7, every other day during hospitalization if hospitalized, week 2 (day 14), and week 4 (day 28).

[0122] As used herein, the "baseline" of cytokines is defined as the last value measured before conditioning chemotherapy.

[0123] When used in this specification, the change in magnification from baseline on day X is:

number

[0124] As used herein, “post-baseline cytokine peak” is defined as the maximum level of serum cytokines achieved from baseline (-5 days) to day 28.

[0125] As used herein, the "time to cytokine peak" after CAR T cell injection is defined as the number of days from day 0 to the day on which the cytokine peak is achieved.

[0126] As used herein, the “Area Under Curve (AUC)” of cytokine levels from day -5 to day 28 is defined as the area under the curve on the plot of cytokine levels against scheduled visits from day -5 to day 28. This AUC represents the total level of cytokines over a long period. Assuming that cytokines and CAR+ T cells are measured at specific, separate time points, the trapezoidal rule may be used to estimate the AUC.

[0127] As used herein, a critically ill adverse event (TEAE) is defined as an adverse event (AE) that occurs during or after the first dose of conditioning chemotherapy. Adverse events may be coded using the Drug Regulatory Terminology Dictionary (MedDRA) version 22.0 and graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. Cytokine release syndrome (CRS) events may be graded at the syndrome level according to Lee et al. (Lee et al, 2014 Blood. 2014;124(2):188-95). Individual CRS symptoms may be graded according to CTCAE 4.03. Neurological events may be identified using a search strategy based on known neurotoxicities associated with CAR T immunotherapy, for example, as described in Topp, MS et al. Lancet Oncology. 2015;16(1):57-66.

[0128] Various aspects of this disclosure are described in more detail in the following subsections. Characterization of serum protein profiles in immunotherapy-treated cancer patients

[0129] In some embodiments, the present disclosure provides methods for characterizing the serum proteomics profile of cancer patients prior to treatment by immunotherapy and / or preconditioning. In one embodiment, the immunotherapy is selected from, among other things, chimeric receptor therapies (e.g., YESCARTA® axi-capbutagen silol-ucel (axi-cel), TECARTUS®-brexcabutagen silol-ucel / KTE-X19, KYMRIAH® (tisagen lecleucel), etc.), TCRs, TILs, and immune checkpoint inhibitors. In one embodiment, the immunotherapy product comprises autologous or allogeneic CAR T cells. In one embodiment, the immunotherapy comprises T cell receptor-modified T cells. 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 characteristics are obtained through a pre-specified set of proteins and analyzed through OPI and machine learning models. In some embodiments, serum levels may be measured by ELISA. In some embodiments, serum protein profiles may be used to predict adverse events related to chimeric receptor therapy (e.g., axicaptagen silolucel (axi-cel)) and adverse events in response to all immunotherapies (e.g., T-cell, non-T-cell, TCR-based therapy, CAR-based therapy, bispecific T-cell engager (BiTE), and / or immune checkpoint blockade).

[0130] In one embodiment, the disclosure provides that baseline (preconditioning) serum levels of certain proteins associated with metabolic processes and leukocyte activation may be positively correlated with and biomarkers of poor prognostic factors for immunotherapy, including international prognostic indicators and baseline systemic tumor tissue volume. In one embodiment, the immunotherapy is T-cell therapy. In some embodiments, the T-cell therapy includes adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation. In a particular embodiment, eACT includes the administration of engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, eACT includes the administration of engineered antigen-specific T-cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is CAR T-cell or TCR T-cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T-cell therapy.

[0131] Accordingly, in one embodiment, the present disclosure provides a method for predicting international prognostic indicators and baseline systemic tumor tissue volume parameters in cancer patients based on baseline (preconditioning) serum levels of metabolic process markers and / or leukocyte activation markers in the patient.

[0132] In one embodiment, the disclosure provides that an increase in the pre-treatment expression level of at least one of CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A correlates with an increased likelihood of response to a cell therapy product in a patient, while an increase in the pre-treatment levels of BNIP3L, MXI1, ADM, PLOD2, P4HA1, ALDOC, SLC2A1, PDK1, P4HA2, BNIP3, NOS2, IL21R, KIR2DL3, KIR3DL1, and KIR3DL2 correlates with a diminished increase in the likelihood of response to a cell therapy product in a patient. In one embodiment, this information is used to make decisions regarding immunotherapy, including whether to administer immunotherapy, whether to administer immunotherapy as a second-line or third-line therapy, what dose of immunotherapy to administer, what dosing regimen to follow, and / or which drugs should be administered to the patient before, after, and / or during immunotherapy administration.

[0133] In one embodiment, high levels of serum biomarkers are at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times higher than the median. In one embodiment, the level of a protein biomarker is high or low if it is 95% to 100% above or below the median or each of the values ​​identified above, such as 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. All listed values ​​may be modified by the term "above".

[0134] In one embodiment, the present disclosure is a method for treating a subject with immunotherapy having a high systemic tumor tissue volume, wherein immune activation-mediated stress in the subject is reduced by administering one or more drugs or treatments that result in reduced inflammation (e.g., lower cytokine induction in the blood), and / or 500-600 mg / m² prior to immunotherapy. 2 Cyclophosphamide and 30 mg / m² per day 2 The present invention provides a method for reducing lymphocyte depletion by using an alternative lymphocyte depletion regimen that does not involve a 3-day administration of fludarabine per day. In one embodiment, baseline systemic tumor tissue volume (SPD) is 2500, 3000, 3500, or 4000 mm³. 2 More preferably, 3000mm 2 A subject has a high systemic tumor tissue volume (as assessed by SPD and / or metabolic tumor volume) if the metabolic tumor volume is greater than and / or exceeds the median of a representative tumor population (e.g., greater than 100 mL or greater than 150 mL).

[0135] In one embodiment, the present disclosure is a method for treating a subject with high international prognostic indicators, 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 500-600 mg / m² prior to immunotherapy. 2 Cyclophosphamide and 30 mg / m² per day 2 The present invention provides a method that reduces the 3-day administration of fludarabine per day by using an alternative lymphocyte depletion regimen that does not involve a 3-day dose of fludarabine per day. In one embodiment, subjects have a high International Prognostic Index (IPI) if the IPI is greater than 1, 2, or 3.

[0136] In one embodiment, the immunotherapy is 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 includes adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), iPSCs, checkpoint inhibitors, and allogeneic T-cell transplantation. In a particular embodiment, the eACT includes the administration of engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. In another embodiment, the eACT includes the administration of engineered antigen-specific T-cell receptor (TCR)-positive (+) T cells. In one embodiment, the immunotherapy is CAR T-cell therapy or TCR T-cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T-cell therapy. Examples of target tumor antigens are enumerated elsewhere in this specification. Examples of cancers that can be treated by the methods of this disclosure are also shown elsewhere in this specification.

[0137] In one embodiment, an agent(s) administered in combination with immunotherapy to activate the immune system and / or reduce endothelial cell destruction, wherein the combination therapy reduces cytokine induction and / or reduces endothelial cell destruction. The agent(s) are selected from anti-IL-1 (e.g., anakinra), T-cell activating inhibitors (e.g., dasatinib), JAK inhibitors (e.g., filgotinib), anti-GM-CSF (e.g., renzilumab), anti-TNF (e.g., infliximab), Ang2 inhibitors (e.g., azilsartan), anti-angiogenic therapies (e.g., bevacizumab), anti-IFNg (e.g., emaparmab-lzsg), ​​etc. In one embodiment, the immunotherapy is administered in combination with a therapy that enhances T-cell proliferation. In one embodiment, the above combination therapy includes treatment with pembrolizumab, lenalidomide, epcolitamab, and utoliumab. In one embodiment, the above therapy includes maglorimab (anti-CD47 antagonist), GSK3745417 (STING agonist), INCB001158 (ARG1 / 2 inhibitor), GS-1423 (CD73xTGFβmAb), sericrelumab (CD40 agonist), GS3583 (FLT3 agonist), pexidartinib (CSF1R inhibitor), epacadostat (IDO1 inhibitor), and GS9620 (TLR agonist).In one embodiment, the drug is selected from: (i) rengilumab; namilumab (AMG203); GSK3196165 / MOR103 / ochilimab (GSK / MorphoSys); KB002 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022 / demicirumab (Morphotek); or any of the biosimilars thereof; E21R; and GM-CSF inhibitors selected from small molecules; (ii) RG7155, PD-0360324, MCS110 / lacnotuzumab, or any of the biosimilar versions thereof; and CSF1 inhibitors selected from small molecules; and / or (iii) GM-CSFR inhibitors, as well as mabrilimumab (formerly CAM-3001; MedImmune, Inc.); kabilizumab (Five Prime Therapeutics); emactuzumab, also known as LY3022855 (IMC-CS4) (Eli Lilly), 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); biosimilar versions of any of these; and small molecules are selected. In some embodiments, additional treatments may be cytokines (e.g., IL-2, IL-15), stimulating antibodies (e.g., anti-41BB, OX-40), checkpoint blockers (e.g., CTLA4, PD-1), or innate immune stimulants (e.g., TLR, STING agonists). In some embodiments, additional therapies may be T-cell mobilization chemokines (e.g., CCL2, CCL1, CCL22, CCL17, and combinations thereof). In some embodiments, additional or multiple therapies are administered systemically or intratumorally. In some embodiments, additional therapies used in combination are administered together with conditioning and / or immunotherapy. In some embodiments, additional therapies used in combination are administered sequentially with conditioning and / or immunotherapy.

[0138] In one embodiment, the drug may / should be administered to the patient before, after, and / or during immunotherapy to reduce grade 3+ CRS in the subject. In one embodiment, the drug(s) are administered to the patient before CAR-T infusion, before the peak of CAR-T proliferation (e.g., days 0-6 after infusion), and / or during the peak of CAR-T proliferation (e.g., days 7-14). In one embodiment, the peak of CAR-T proliferation is days 7-14 after infusion. In one embodiment, the peak of CAR-T proliferation is days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 after infusion. In one embodiment, the period after CAR-T peak proliferation is the period from day 14 to day 28 after infusion. In one embodiment, the period after CAR-T peak proliferation is days 1-5, days 5-10, days 10-15, days 15-20, days 20-25; and days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, and any other day.

[0139] In one embodiment, immunotherapy is combined with low-dose radiation, enhancement of T-cell activity by immune checkpoint blockers, and / or T-cell agonists. In one embodiment, the T-cell agonist is selected from pembrolizumab, lenalidomide, epcolitamab, and utoliumab. In one embodiment, the combination agent is selected from checkpoint inhibitors (e.g., anti-PD1 antibodies such as pembrolizumab (Keytruda), cemiprimab (Libtayo), and nivolumab (Opdivo); anti-PD-L1 antibodies such as atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi); and / or anti-CTLA-4 antibody such as ipilimumab (Yervoy)).

[0140] In one embodiment, the preconditioning regimen is a lymphocyte apheresis regimen. In one embodiment, the lymphocyte apheresis therapy regimen(s) is selected from one of several possible regimens of cyclophosphamide / fludarabine, bendamustine, whole-body irradiation, anti-CD45 (apamistamab), and other chemotherapeutic agents (e.g., AVM0703, busulfan, thiotepa / etoposide, pentostatin). Further conditioning methods and regimens can be found elsewhere in this specification.

[0141] In one embodiment, the present disclosure provides a method for improving immunotherapy (e.g., CAR T-cell therapy) by optimizing bridging therapy to modulate the tumor microenvironment to a more favorable immune tolerance state. In one embodiment, the optimization includes administering bridging therapy with an immunomodulatory imide drug (IMID) / cereblon modulator (e.g., lenalidomide, pomalidomide, iverdide, and apremilast). In one embodiment, the optimization includes administering bridging therapy by local radiation therapy.

[0142] In one embodiment, the disclosure provides a method for improving immunotherapy (e.g., CAR T-cell therapy) by optimizing a bridging therapy to reduce systemic tumor tissue volume before administration of the immunotherapy (e.g., CAR T-cell therapy). In one embodiment, the optimization includes administering a bridging therapy with R-CHOP, bendamustine, an alkylating agent, and / or a platinum-based agent. Other exemplary bridging therapies are described elsewhere in this application.

[0143] In one embodiment, the disclosure provides a method for improving immunotherapy (e.g., CAR T-cell therapy) by optimizing conditioning therapy to modulate the tumor microenvironment to a more favorable immune tolerance state (e.g., less myeloid inflammation in the tumor mesenteric artery). In one embodiment, the optimization includes adding local radiation to conditioning with cyclophosphamide / fludarabine. In one embodiment, the optimization includes administering a platinum-based agent as the conditioning agent.

[0144] In one embodiment, the present disclosure provides a method for improving immunotherapy (e.g., CAR T cell therapy) by co-administering a biological response modifier concurrently with or after the administration of the immunotherapy (e.g., CAR T cell therapy) to activate CAR T cell activity. In one embodiment, the method includes the administration of gamma chain cytokines (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). In one embodiment, the method includes the administration of a checkpoint blocker (e.g., anti-CTLA-4).

[0145] In one embodiment, the disclosure provides a method for improving immunotherapy (e.g., CAR T-cell therapy) by reprogramming T cells to overcome a detrimental tumor microenvironment, including a low T / M ratio, high systemic tumor tissue volume, high TME myeloid cell density, and / or high TME myeloid inflammation levels. In one embodiment, 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 inductive promoter.

[0146] In one embodiment, the disclosure provides a method to improve CAR T cell therapy by optimizing T cell production to facilitate CAR T cells overcoming a potentially harmful tumor microenvironment, the characteristics of which the tumor microenvironment may include a low T / M ratio, high systemic tumor tissue volume, high TME myeloid cell density, and / or high TME myeloid inflammation levels. In one embodiment, the characteristics of which the TME may be harmful include a low T / M ratio (in the range of -0.5 to 4) and high systemic tumor tissue volume (3000 to 40000 mm²).2 within the range), high myeloid cell density (1000 - 4000 cells / mm 2 within the range), and / or high TME myeloid inflammation level (within the range of 27 - 2000). In one embodiment, the method includes engineering CAR T cells to express a gamma chain receptor cytokine. In one embodiment, the gamma chain receptor cytokine is expressed under a constitutive promoter or an inducible promoter. In one embodiment, the method includes expanding T cells in the presence of a gamma chain cytokine such as IL-15.

[0147] Clinical outcome In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is a durable response. In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is no response. In some embodiments, the clinical outcome is a partial response. In some embodiments, the clinical outcome is an objective response. In some embodiments, the clinical outcome is survival. In some embodiments, the clinical outcome is recurrence.

[0148] In some embodiments, the objective response (OR) is determined according to the revised version of the IWG lymphoma response criteria (Cheson, 2007) and is determined by the IWG lymphoma response criteria (Cheson et al. Journal of Clinical Oncology 32, no. 27 (September 2014) 3059 - 3067). The duration of the response is evaluated. The progression-free survival (PFS) is evaluated by the investigator's assessment according to the Lugano classification response criteria.

[0149] In some embodiments, part of the clinical outcome is the evaluation of adverse events. In this regard, CRS grading is performed 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 was assessed by monitoring the patient for signs and symptoms of neurotoxicity by excluding other causes of neurological symptoms. Patients experiencing neurotoxicity of grade 2 or higher should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

[0150] In some embodiments, the method includes monitoring the patient daily for at least 7 days after injection at an accredited medical facility for signs and symptoms of neurotoxicity. In some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity for 4 weeks after injection.

[0151] In some embodiments, neurotoxic symptoms are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, adverse reaction symptoms are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are instructed to stay near an accredited medical facility for at least four weeks after infusion.

[0152] The clinical outcomes of CAR T cell therapy depend on the level of CAR T cells in the blood. In some embodiments, response, blood levels of CAR T cells, or immune-related factors are determined by follow-up approximately 1, 2, 3, 4, 5, 6, or 7 days after administration of engineered CAR T cells. In some embodiments, response, blood levels of CAR T cells, or immune-related factors are determined by follow-up approximately 1, 2, 3, or 4 weeks after administration of engineered CAR T cells. In some embodiments, response, blood levels of CAR T cells, and / or immune-related factors are determined by follow-up approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months after administration of the engineered CAR T cells. In some embodiments, response, blood levels of CAR T cells, and / or immune-related factors are determined by follow-up approximately 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, or 5 years after administration of the engineered CAR T cells.

[0153] In some embodiments, the methods described herein may provide clinical efficacy. 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 achieve clinical efficacy. In some embodiments, approximately 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% of patients, and any unlisted percentage in between, achieve clinical efficacy. In some embodiments, the success rates are 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 any other percentage not listed, and in the range of 1% to 100%. In some embodiments, the response rate is 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 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 various other ranges from 95% to 100%. Chimeric antigen receptor

[0154] 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 in immune cells, including T cells and other lymphocytes, according to techniques known in the art. With CARs, a single receptor can be programmed to both recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells possessing that antigen. If these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to enhance their potency. See U.S. Patents No. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (cited above), 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).

[0155] In some embodiments, the co-stimulatory domain, which includes a truncated hinge domain ("THD"), further comprises some or all members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

[0156] In some embodiments, the THD is derived from the human complete hinge domain ("CHD"). In other embodiments, the THD is derived from the CHD of a rodent, mouse, or primate (e.g., non-human primate) co-stimulatory protein. In some embodiments, the THD is derived from a chimeric CHD of the co-stimulatory protein.

[0157] The co-stimulatory domains of the CARs of this disclosure may further include a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be fused to the extracellular domain of the CAR. The co-stimulatory domain may similarly be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally binds to one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex by avoiding binding to transmembrane domains of the same or different surface membrane proteins. The transmembrane domain may originate from either a natural or synthetic source. If from a natural source, the domain may originate from any membrane-binding protein or transmembrane protein.Membrane-spanning regions that are particularly useful in the present disclosure can be derived from any membrane-bound protein or transmembrane protein such as 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell 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 fragments, cleavages, or combinations thereof.

[0158] Optionally, a short linker may form a bond between one or more of the extracellular, transmembrane, and intracellular domains of the CAR. The linkers described herein may be used as peptide tags. The linker peptide sequence may be of any length suitable for linking one or more proteins of interest, and is preferably designed to be sufficiently flexible to allow proper folding and / or function and / or activity of one or both of the peptides it links. Thus, the linker peptide may have a length of 10 amino acids or less, 11 amino acids or less, 12 amino acids or less, 13 amino acids or less, 14 amino acids or less, 15 amino acids or less, 16 amino acids or less, 17 amino acids or less, 18 amino acids or less, 19 amino acids or less, or 20 amino acids or less. In some embodiments, the linker peptide is 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 contains at least 7 and 20 amino acids, at least 7 and 19 amino acids, at least 7 and 18 amino acids, at least 7 and 17 amino acids, at least 7 and 16 amino acids, at least 7 and 15 amino acids, at least 7 and 14 amino acids, at least 7 and 13 amino acids, at least 7 and 12 amino acids, or at least 7 and 11 amino acids. In certain embodiments, the linker contains 15 to 17 amino acids, and in certain embodiments, 16 amino acids. In some embodiments, the linker contains 10 to 20 amino acids. In some embodiments, the linker contains 14 to 19 amino acids. In some embodiments, the linker contains 15 to 17 amino acids. In some embodiments, the linker contains 15 to 16 amino acids. In some embodiments, the linker contains 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.

[0159] 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, to control expression by addition of a small molecule, the spacer domain may include a chemically inducible dimerization factor. In some embodiments, no spacer is used.

[0160] The intracellular (signaling) domain of the engineered T cells of the disclosure can result in signal transduction to an activation domain, which then activates at least one of the normal effector functions of immune cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity that includes secretion of cytokines.

[0161] In certain embodiments, preferred intracellular signaling domains include, but are not limited to, 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB, KIRDS2, LAT, ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class I molecule, N This includes, but is not limited to, KG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death 1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76), TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage, or combinations thereof. antigen binding molecule

[0162] Suitable CARs and TCRs can bind to antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with their target antigen. In some embodiments, the antigen-binding molecule is an antibody fragment, for example, one or more single-chain antibody fragments ("scFv"). An scFv is a single-chain antibody fragment having variable regions of linked antibody heavy and light chains. See U.S. Patents No. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. The scFv retains the ability of the parent antibody to specifically interact with the target antigen. Since scFv can be manipulated to be expressed as part of a single chain along with other CAR components, they are useful for chimeric antigen receptors. See also Id. Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. Antigen-binding molecules are typically contained in the extracellular portion of the CAR or TCR so that they can recognize and bind to the antigen of interest. Bispecific and multispecific CARs and TCRs having specificity for two or more targets of interest are intended to be within the scope of this disclosure.

[0163] In some embodiments, the polynucleotide encodes a CAR or TCR comprising a (shortened) 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 a tumor-associated surface antigen, e.g., 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, β-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 antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M Endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-related protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-related antigen, sphingoglycolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-related antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGF-II receptor, IGF-II, IL-11Rα, IL-13Rα2, influenza virus-specific antigen;CD38, insulin growth factor (IGFl)-1, intestinal carboxylesterase, κ chain, LAGA-1a, λ chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific antigen or tissue-specific antigen, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecule, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA Selected from IX, MUC-1, variant hsp70-2, variant p53, variant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivorin and telomerase, TAG-72, extradomain A (EDA) and extradomain B (EDB) of fibronectin, and A1 domain of tenascin C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens, e.g., HIV-specific antigen (e.g., HIV gp120), and any derivatives or variants of these surface antigens. Manipulated T cells and products

[0164] In one embodiment, immunotherapy is T-cell therapy. In some embodiments, donor T cells for use in T-cell therapy are obtained from a patient (for example, for autologous T-cell therapy). In other embodiments, donor T cells for use in T-cell therapy are obtained from a subject that is not a patient. In certain embodiments, T cells are tumor-infiltrating lymphocytes (TILs), modified autologous T cells (eACT®), allogeneic T cells, heterologous T cells, or any combination thereof. In some embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient with cancer or a tumor. In some embodiments, the donor subject is a human patient who does not have cancer or a tumor.

[0165] 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 from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, or tumors, or differentiated in vitro. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a suitable buffer or medium for further processing. In some embodiments, the cells are washed with PBS. As understood, the washing step can be used, for example, by using a semi-automatic flow-through centrifuge, e.g., Cobe® 2991 cell processing device, Baxter CytoMate®, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or in other salines, with or without buffers. In some embodiments, undesirable components are removed from the apheresis sample. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0166] In some embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes, for example, using centrifugation with a PERCOLL® gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the Art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. In some embodiments, cell sorting and / or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate the desired cell population for use in the present disclosure.

[0167] In some embodiments, PBMCs are used directly for genetic modification (such as CAR) of immune cells using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated, and both cytotoxic T lymphocytes and helper T lymphocytes are sorted into subpopulations of naive T cells, memory T cells, and effector T cells before or after genetic modification and / or proliferation.

[0168] In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, phenotypic markers for central memory T cells include the expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127, as well as negativity for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens.

[0169] In some embodiments, immune cells, such as T cells, are genetically modified (manipulated) after isolation using known methods, or the immune cells are activated and proliferated in vitro (or differentiated in the case of progenitor cells) before genetic modification. In other embodiments, immune cells, such as T cells, are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), and then activated and / or proliferated in vitro. Methods for activating and proliferating T cells are known in the art and, as non-limiting examples, are described in U.S. Patents 6,905,874, 6,867,041 and 6,797,514, and International Publications 2015 / 20096, 2016 / 191756, 2016 / 191755, 2019 / 079564 and 2021 / 092290, each of which is incorporated herein by reference in its entirety. Their contents are incorporated herein by reference in their entirety. Generally, such methods involve contacting PBMCs or isolated T cells with stimulants and co-stimulants, generally bound to beads or other surfaces, such as anti-CD3 and anti-CD28 antibodies, in a culture medium containing a suitable cytokine such as IL-2. The anti-CD3 and anti-CD28 antibodies bound to the same beads function as “surrogate” antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to proliferate using feeder cells and appropriate antibodies and cytokines, such as in the methods described in U.S. Patent No. 6,040,177 and No. 5,827,642 and International Publication No. 2012 / 129514 (the contents of which are incorporated herein by reference in their entirety).

[0170] In some embodiments, the composition containing the manipulated T cells includes a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition includes excipients.

[0171] In some embodiments, the composition is selected for parenteral delivery, inhalation, or delivery via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In some embodiments, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8. In some embodiments, when parenteral administration is intended, the composition is in the form of a pyrogenically-free parenterally acceptable aqueous solution containing the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In some embodiments, the parenteral injection vehicle is sterile distilled water, in which the composition described herein is formulated as a sterile isotonic solution with or without at least one additional therapeutic agent and is appropriately stored. In some embodiments, the preparation involves formulation of the molecule of interest with a polymer compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, which is then delivered by accumulation injection. In some embodiments, the molecule of interest may be introduced using an implantable drug delivery device.

[0172] In some embodiments, the manipulated T cells are administered in a therapeutically effective dose. For example, the therapeutically effective dose of manipulated T cells is at least about 10 4 Individual cells, at least about 10 5 Individual cells, at least about 10 6 Individual cells, at least about 10 7 Individual cells, at least about 10 8 Individual cells, at least about 10 9 pieces, or at least about 10 10 It may be a number. In another embodiment, the therapeutically effective amount of T cells is about 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of T cells is approximately 2 × 10⁻⁶ 6Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 4×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 6×10 6 Individual cells / kg, approximately 7×10 6 Individual cells / kg, approximately 8×10 6 Individual cells / kg, approximately 9×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 4×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 6×10 7 Individual cells / kg, approximately 7×10 7 Individual cells / kg, approximately 8×10 7 Individual cells / kg, or approximately 9 × 10⁻⁶ cells 7 It is expressed as individual cells / kg.

[0173] In some embodiments, the therapeutically effective dose of manipulated viable T cells is approximately 1 × 10⁶ per kg of body weight. 6 pieces and approximately 2 × 10 6 From individual manipulated surviving T cells, approximately 1 × 10⁶ cells 8 This is until the maximum dose of individual manipulated viable T cells is reached.

[0174] In some embodiments, the engineered T cells are anti-CD19 CAR T cells. In some embodiments, the anti-CD19 CAR T cells are axicapbutagen silolucel products, YESCARTA® axicapbutagen silolucel (axi-cel), TECARTUS®-brexcapbutagen autolucel / KTE-X19, KYMRIAH® (tisagenlecleucel), lysocabbutagen maralucel. In some embodiments, the engineered T cells are anti-BCMA CAR T cells such as idekabutagen bicleucel / bb2121. In some embodiments, the product meets commercial standards. In some embodiments, the product does not meet commercial standards (off-spec product, OOS). In some embodiments, the OOS product has fewer, more undifferentiated CCR7+ T cells compared to axicapbutagen silolucel products that meet commercial standards. N and T CM , as well as a higher proportion of more differentiated CCR7-T EM +T EFF The product contains cells. In some embodiments, the OOS product resulted in lower median CAR T cell peak levels after administration compared to the commercially available product. In some embodiments, the OOS product still demonstrated a manageable safety profile and significant clinical efficacy.

[0175] This application also provides doses and administrations of cells prepared by the method of this application, for example, an infusion bag for CD19-directed genetically modified autologous T cell immunotherapy containing approximately 68 mL of chimeric antigen receptor (CAR)-positive T cell suspension for infusion. In some embodiments, the CAR T cells are formulated in approximately 40 mL for infusion. In some embodiments, the CAR T cell product is formulated in total volumes of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900, and 1000 mL. In one embodiment, the doses and administrations of cells prepared by the method of this application, for example, an infusion bag for CD19-directed genetically modified autologous T cell immunotherapy, contain 1 × 10⁶ cells in approximately 40 mL. 6Contains a suspension of CAR-T positive cells. The target dose is approximately 1 × 10⁶ cells per kg of body weight. 6 ~about 2×10 6 These may be CAR-positive surviving T cells, up to 2 × 10⁶ 8 These could be CAR-positive surviving T cells.

[0176] In some embodiments, the dosage form comprises a cell suspension for infusion in a single-dose patient-specific infusion bag, the route of administration is intravenous, and the entire contents of each single-dose patient-specific bag are infused over 30 minutes by gravity or a peristaltic pump. In one embodiment, the administration regimen is 2.0 × 10 6 Individual anti-CD19 CAR T cells / kg body weight (±20%), maximum dose 2 × 10⁶ 8 This is a single infusion consisting of 100 anti-CD19 CAR T cells (for a subject weighing ≥100 kg). In some embodiments, the T cells constituting the dose are CD19 CAR-T cells. Conditioning agent

[0177] In some embodiments, the subject is administered a conditioning agent before immunotherapy. In some embodiments, conditioning is performed by radiation therapy. In some embodiments, the conditioning therapy is lymphocyte apheresis chemotherapy.

[0178] In one embodiment, the conditioning therapy comprises an alkylating agent selected from the group consisting of melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uracil mustard, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, alkyl sulfonates, busulfan, thiotepa or its analogs, 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 conditioning agent selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.

[0179] In another embodiment, one or more conditioning agents can comprise a platinum-based chemotherapeutic agent. In certain embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, any analogs or functional derivatives thereof, and any combination thereof.

[0180] In another embodiment, one or more 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 analogs or functional derivatives thereof, and any combination thereof. In one embodiment, one or more conditioning agents comprise fludarabine.

[0181] In some embodiments, one or more preconditioning agents may include cyclophosphamide and purine analogs. Purine analogs can be selected from the group consisting of azathioprine, 6-mercaptopurine, mercaptopurine, thiopurine, thioguanine, fludarabine, pentostatin, cladribine, any analogs or functional derivatives thereof, and any combination thereof. In a particular embodiment, one or more preconditioning agents include cyclophosphamide and pentostatin. In a particular embodiment, one or more preconditioning agents include cyclophosphamide and fludarabine. As non-limiting examples, dosages and regimens of cyclophosphamide and fludarabine are described in at least International Publication No. 2019 / 079564, International Publication No. 2021 / 092290, International Publication No. 2015 / 20096 and International Publication No. 2016 / 191755, each of which is incorporated herein by reference in whole.

[0182] In certain embodiments, a first dose (also applicable to repeated doses) of one or more preconditioning agents is administered to the patient. For example, in some embodiments, the first dose of cyclophosphamide is approximately 300 mg / m². 2 / day~about 2000mg / m 2 This is / day. In another embodiment, the first dose of cyclophosphamide is 300 mg / m². 2 Higher than / day, 2000 mg / m² 2 Lower than / day. In other embodiments, the dose of cyclophosphamide is approximately 350 mg / m². 2 / day~about 2000mg / m 2 / day, at least approximately 400 mg / m² 2 / day~about 2000mg / m 2 / day, about 450mg / m 2 / day~about 2000mg / m 2 / day, about 500mg / m 2 / day~about 2000mg / m 2 / day, about 550mg / m 2 / day~about 2000mg / m2 / day, or about 600 mg / m 2 / day to about 2000 mg / m 2 / day. In other embodiments, the dosage 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 dosage 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 / m 2 / day, about 650 mg / m 2 / day, about 700 mg / m 2 / day, about 800 mg / m 2 / day, about 900 mg / m 2 / day, or about 1000 mg / m 2 / day.

[0183] In other embodiments, the first dosage of cyclophosphamide (also applicable to the repeated dosage) is about 200 mg / m 2 / day to about 3000 mg / m 2 / day. In another embodiment, the first dosage of cyclophosphamide is higher than 200 mg / m 2 / day and lower than 3000 mg / m 2 / day. In other embodiments, the dosage of cyclophosphamide is about 200 mg / m 2 / day to about 3000 mg / m2 / day, approximately 300mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 400mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 500mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 600mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 700mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 800mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 900mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1000mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1100mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1200mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1300mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1400mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1500mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1600mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1700mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1800mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 1900mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 2000mg / m 2 / Daily ~ Approximately 3000mg / m 2 / day, approximately 200mg / m 2 / Daily ~ Approximately 2900mg / m 2 / day, approximately 400mg / m 2 / Daily ~ Approximately 2800mg / m 2 / day, about 500mg / m 2 / day ~ approx. 2700mg / m 2 / day, about 600mg / m 2 / day ~ approx. 2600mg / m 2 / day, about 700mg / m 2 / day~about 2500mg / m 2 / day, about 800mg / m 2 / day ~ approx. 2400mg / m 2 / day, approximately 900mg / m 2 / day ~ approx. 2350mg / m 2 / day, about 1000mg / m 2 / day ~ approx. 2300mg / m 2 / day, about 1100mg / m 2 / day ~ approx. 2250mg / m 2 / day, or approximately 1100 mg / m² 2 / day ~ approx. 2220mg / m 2 This is / day. In one embodiment, the first dose of cyclophosphamide is 200 mg / m². 2 This is / day. In another embodiment, the first dose of cyclophosphamide is 300 mg / m². 2 This is / day. In another embodiment, the first dose of cyclophosphamide is 500 mg / m². 2 / day

[0184] In some embodiments, the first dose of fludarabine (also applicable to repeated doses) is approximately 20 mg / m². 2 / day~about 900mg / m 2 The dose is 30 mg / m² / day. In some embodiments, the dose of fludarabine is 30 mg / m². 2 Higher than / day, 900mg / m² 2 Lower than / day. In some embodiments, the dose of fludarabine is approximately 35 mg / m². 2 / day~about 900mg / m 2 / day, about 40mg / m 2 / day~about 900mg / m 2 / day, about 45mg / m 2 / day~about 900mg / m 2 / day, about 50mg / m 2 / day~about 900mg / m 2 / day, about 55mg / m 2 / day~about 900mg / m 2 / day, or approximately 60 mg / m² 2 / day~about 900mg / m 2 The dose is approximately 35 mg / m² / day. In some embodiments, the dose of fludarabine is approximately 35 mg / m². 2 / day~about 900mg / m 2 / day, about 35mg / m 2 / day~about 800mg / m 2 / day, about 35mg / m 2 / day~about 700mg / m 2 / day, about 35mg / m 2 / day~about 600mg / m 2 / day, about 35mg / m 2 / day~about 500mg / m 2 / day, about 35mg / m 2 / day ~ approx. 400mg / m 2 / day, about 35mg / m 2 / day ~ approx. 300mg / m 2 / day, about 35mg / m 2 / day~about 200mg / m 2 / day, about 35mg / m 2 / day~about 100mg / m 2 / day, about 40mg / m 2 / day~about 90mg / m 2 / day, about 45mg / m 2 / day~about 80mg / m 2 / day, about 45mg / m 2 / day~about 70mg / m 2 / day, or approximately 50 mg / m² 2 / day~about 60mg / m 2 The dose is approximately 20 mg / m² / day. In some embodiments, the dose of fludarabine is approximately 20 mg / m². 2 / day, about 25mg / m 2 / day, about 30mg / m 2 / day, about 35mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 / day, about 65mg / m 2 / day, about 70mg / m 2 / day, about 75mg / m2 / day, about 80mg / m 2 / day, about 85mg / m 2 / day, about 90mg / m 2 / day, approximately 95mg / m 2 / day, about 100mg / m 2 / day, about 200mg / m 2 / day, or approximately 300 mg / m² 2 The dose is approximately 20 mg / m² / day. In some embodiments, the dose of fludarabine is approximately 20 mg / m². 2 / day, about 25mg / m 2 / day, about 30mg / m 2 / day, about 35mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 / day, about 65mg / m 2 / day, about 70mg / m 2 / day, about 75mg / m 2 / day, about 80mg / m 2 / day, about 85mg / m 2 / day, about 90mg / m 2 / day, approximately 95mg / m 2 / day, or approximately 100 mg / m² 2 In other embodiments, the dose of fludarabine is approximately 110 mg / m². 2 / day, 120mg / m 2 / day, 130mg / m 2 / day, 140mg / m 2 / day, 150mg / m 2 / day, 160mg / m 2 / day, 170mg / m 2 / day, 180mg / m 2 / day, or 190 mg / m² 2 The dose is approximately 210 mg / m² / day. In some embodiments, 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 290 mg / m² 2 The dose is / day. In one particular embodiment, the dose of fludarabine is approximately 20 mg / m². 2 The dose is / day. In one particular embodiment, the dose of fludarabine is approximately 25 mg / m². 2 This is / day. In another embodiment, the dose of fludarabine is approximately 30 mg / m². 2 This is / day. In another embodiment, the dose of fludarabine is approximately 60 mg / m². 2 / day

[0185] The timing of administration of one or more preconditioning agents can be adjusted to maximize their effect. In certain embodiments, one or more preconditioning agents include two or more preconditioning agents. The two or more preconditioning agents can be administered simultaneously or sequentially. In a particular embodiment, a first preconditioning agent, for example, cyclophosphamide, is administered to the patient before or after a second preconditioning agent, for example, fludarabine.

[0186] 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 The daily dose is 20 mg / m² of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 300 mg / m². 2 The daily dose is 30 mg / m², and the fludarabine dose is 30 mg / m². 2 In other embodiments, the dose of cyclophosphamide is 300 mg / m². 2 The daily dose is 60 mg / m² of fludarabine.2 In other embodiments, the dose of cyclophosphamide is 500 mg / m². 2 The daily dose is 20 mg / m² of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 500 mg / m². 2 The daily dose is 30 mg / m², and the fludarabine dose is 30 mg / m². 2 In other embodiments, the dose of cyclophosphamide is 500 mg / m². 2 The daily dose is 60 mg / m² of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m². 2 The daily dose is 20 mg / m² of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m². 2 The daily dose is 30 mg / m², and the fludarabine dose is 30 mg / m². 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m². 2 The daily dose is 60 mg / m² of fludarabine. 2 / day

[0187] As described herein, the day on which T-cell therapy is administered is designated as day 0. One or more preconditioning agents may be administered at any point prior to the administration of T-cell therapy. In some embodiments, the administration of one or more preconditioning agents is initiated at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day before the administration of T-cell therapy. In other embodiments, the administration of one or more preconditioning agents is initiated at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days before the administration of T-cell therapy. In one embodiment, the administration of one or more preconditioning agents is initiated about 7 days before the administration of T-cell therapy. In another embodiment, the administration of one or more preconditioning agents is initiated about 5 days before the administration of T-cell therapy.

[0188] In one embodiment, the administration of the first preconditioning agent is started approximately 7 days before the administration of T-cell therapy, and the administration of the second preconditioning agent is started approximately 5 days before the administration of T-cell therapy. In a particular embodiment, the first preconditioning agent is administered to the patient for 2 days, approximately 7 days and approximately 6 days before the administration of T-cell therapy. In another embodiment, the second preconditioning agent is administered to the patient for 5 days, approximately 5 days, 4 days, 3 days, 2 days and 1 day before the administration of T-cell therapy. In yet another embodiment, the first preconditioning agent is administered to the patient for 3 days, approximately 5 days, 4 days and 3 days before the administration of T-cell therapy.

[0189] In one particular embodiment, administration of cyclophosphamide is initiated approximately 7 days before administration of T-cell therapy, and administration of a purine analog (e.g., fludarabine or pentostatin) is initiated approximately 5 days before administration of T-cell therapy. In another embodiment, administration of cyclophosphamide is initiated approximately 5 days before administration of T-cell therapy, and administration of a purine analog (e.g., fludarabine or pentostatin) is initiated approximately 5 days before administration of T-cell therapy.

[0190] The timing of administration of each component can be adjusted to maximize its effect. Generally, one or more preconditioning agents can be administered daily. In some embodiments, one or more preconditioning agents are administered daily for about 2, 3, 4, 5, 6, or 7 days. In some embodiments, one or more preconditioning agents can be administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days. In a particular embodiment, one or more preconditioning agents are administered daily for about 3 days.

[0191] As described herein, the day on which T-cell therapy is administered to a patient is designated as day 0. In some embodiments, one or more preconditioning agents, such as cyclophosphamide, are administered to the patient on days 7 and 6 prior to day 0 (i.e., day -7 and day -6). In other embodiments, one or more preconditioning agents, such as cyclophosphamide, are administered to the patient on day -5, day -4, and day -3. In some embodiments, one or more preconditioning agents, such as fludarabine, are administered to the patient on day -5, day -4, day -3, day -2, and day -1. In other embodiments, one or more preconditioning agents, such as fludarabine, are administered to the patient on day -5, day -4, and day -3.

[0192] One or more preconditioning agents, such as cyclophosphamide and fludarabine, may be administered on the same day or on different days. If cyclophosphamide and fludarabine are administered on the same day, the cyclophosphamide dose may be administered either before or after the fludarabine dose. In one embodiment, the cyclophosphamide dose is administered to the patient on days -7 and -6, and the fludarabine dose is administered to the patient on days -5, -4, -3, -2, and -1. In another embodiment, the cyclophosphamide dose is administered to the patient on days -5, -4, and -3, and the fludarabine dose is administered to the patient on days -5, -4, and -3.

[0193] In certain embodiments, one or more preconditioning agents, such as cyclophosphamide and fludarabine, may 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.

[0194] Routes and regimens for administering one or more preconditioning agents are publicly known in the art and are described, for example, in International Publication No. 2019 / 079564, International Publication No. 2021 / 092290, International Publication No. 2015 / 20096 and International Publication No. 2016 / 191755, each of which is incorporated herein by reference in whole. cancer

[0195] The methods disclosed herein may be used to treat cancer in a subject, to reduce tumor size, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to remove a tumor from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0196] Cancers that can be treated include non-angiogenic tumors, tumors that are not yet substantially angiogenic, or tumors that are angiogenic. Cancers may also include solid or non-solid tumors. In some embodiments, cancer is a hematological cancer. In some embodiments, cancer is a leukocyte cancer. In other embodiments, cancer is a plasma cell cancer. In some embodiments, cancer is leukemia, lymphoma, or myeloma. In some embodiments, cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute lymphoblastic leukemia (ALL), and hemophagocytic lymphohistiocytosis (HLH), B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic or acute granulomatous disease, chronic or This includes acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), pilocytic cell leukemia, macrophage activation syndrome (MAS), Hodgkin's disease, large cell granuloma, leukocytosis, lymphoproliferative malignancy, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal hypergammaglobulinemia of unknown significance (MGUS), multiple myeloma, and spinal cord lymphoma. Myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), plasmacytoproliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (e.g., plasmacytoproliferative disorder, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple myeloma), POEMS syndrome (Cro w-Fukase syndrome, Takatsuki disease, PEP syndrome), 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 lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenström macroglobulinemia, DLBCL arising from FL, high-grade B-cell lymphoma, or combinations thereof.

[0197] In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the cancer is relapsed or refractory large B-cell lymphoma (possibly after two or more systemic therapies), including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, 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 systemic therapies), or relapsed or refractory mantle cell lymphoma (MCL).

[0198] In some embodiments, the cancer is non-Hodgkin lymphoma. In some embodiments, the cancer is relapsed / refractory NHL. In some embodiments, the cancer is mantle cell lymphoma.

[0199] In some embodiments, the cancer is advanced-stage low-grade non-Hodgkin lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL). In some embodiments, the patient developed relapsed / treatment-resistant disease after two or more prior treatments, including combinations of anti-CD20 monoclonal antibodies and alkylating agents. In some embodiments, the patient may have been administered PI3K inhibitors. In some embodiments, the patient may have also undergone autologous stem cell transplantation. In some embodiments, the patient undergoes leukocyte apheresis to obtain T cells for CAR T cell production, followed by administration of 500 mg / m² on days -5, -4, and -3. 2 Cyclophosphamide and 30 mg / m² per day 2 The patient received conditioning chemotherapy with fludarabine / day, and on day 0, the patient received 2 × 10 6A single intravenous infusion of CAR T-cell therapy (e.g., axicaptagen silolucel, brexcabutagen autolucel) at a target dose of CAR T cells / kg may be administered. In some embodiments, additional infusions may be provided in subsequent periods. In some embodiments, if the patient progresses after responding at the 3-month evaluation following the initial administration, the patient may be re-treated with CAR T-cell therapy (e.g., axicaptagen silolucel, brexcabutagen autolucel). In some embodiments, the patient may receive bridging therapy. Examples of bridging therapy are presented elsewhere in this specification, including in the examples. In some embodiments, the patient experiences CRS. In some embodiments, CRS is managed using one of the protocols described in this application, including in the examples. In some embodiments, CRS is managed with tocilizumab, corticosteroids, and / or vasopressors.

[0200] In some embodiments, the cancer is relapsed / refractory low-grade non-Hodgkin lymphoma, and the method of treating a subject requiring treatment comprises administering a therapeutically effective dose of CAR T cells to the subject as retreatment, wherein the subject has previously received initial treatment with CAR T cells. In some embodiments, the initial treatment with CAR T cells may be administered as first-line or second-line therapy, wherein optionally, the lymphoma is R / R follicular lymphoma (FL) or marginal zone lymphoma (MZL), and optionally, prior treatment comprises an anti-CD20 monoclonal antibody in combination with an alkylating agent. In some embodiments, the conditioning therapy comprises fludarabine 30 mg / m² on days -5, -4, and -3. 2 IV infusion and cyclophosphamide 500 mg / m² 2 This includes IV injection. In some embodiments, CAR T cell therapy involves 2 × 10⁶ cells on day 0. 6 This involves a single IV infusion of 10 CAR T cells / kg. In some embodiments, at least about 10 4 Individual cells, at least about 10 5 Individual cells, at least about 10 6Individual cells, at least about 10 7 Individual cells, at least about 10 8 Individual cells, at least about 10 9 pieces, or at least about 10 10 A number of CAR T cells are administered. In another embodiment, the therapeutically effective dose of T cells is approximately 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of T cells is approximately 2 × 10⁻⁶ 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 4×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 6×10 6 Individual cells / kg, approximately 7×10 6 Individual cells / kg, approximately 8×10 6 Individual cells / kg, approximately 9×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 4×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 6×10 7 Individual cells / kg, approximately 7×10 7 Individual cells / kg, approximately 8×10 7 Individual cells / kg, or approximately 9 × 10⁻⁶ cells 7 The cell count is individual cells / kg. In some embodiments, the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cells are axicaptagensilolucel CAR T cells. In some embodiments, eligibility criteria for retreatment include a complete response (CR) or partial response (PR) at the 3-month disease evaluation and subsequent progression; no evidence of CD19 disappearance on post-progression biopsy by local re-examination; and / or no grade 4 CRS, neurological events, or life-threatening toxicity resulting from the initial treatment with CAR T cells. In some embodiments, the treatment method is a treatment method followed by a clinical trial (NCT03105336).

[0201] In some embodiments, the cancer is NHL and immunotherapy (e.g., CAR T or TCR T cell therapy) is administered as first-line treatment. In some embodiments, the cancer is LBCL. In some embodiments, the LBCL is high-risk / high-grade LBCL with MYC and BCL2 translocation and / or BCL6 translocation or DLBCL exhibiting an IPI score of 3 or higher at any point before registration. In some embodiments, the first-line treatment includes CAR T cell therapy in combination with a regimen containing an anti-CD20 monoclonal antibody and anthracycline. In some embodiments, the CAR T cell therapy is administered first. In some embodiments, the regimen containing an anti-CD20 monoclonal antibody / anthracycline is administered first. In some embodiments, the treatments are administered at intervals of 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, and less than 1 year. In some embodiments, the method further includes bridging therapy administered after leukocyte apheresis and completed before initiating conditioning chemotherapy. In some embodiments, additional inclusion criteria include age 18 years or older and ECOG PS of 0–1. In some embodiments, the conditioning therapy includes IV infusions of fludarabine 30 mg / m2 and cyclophosphamide 500 mg / m2 on days -5, -4, and -3. Other exemplary beneficial preconditioning treatment regimens are described in U.S. Provisional Patent Applications 62 / 262,143 and 62 / 167,750 and U.S. Patents 9,855,298 and 10,322,146, which are incorporated herein by reference in their entirety. These include, for example, specified beneficial doses of cyclophosphamide (200 mg / m2). 2 / day~2000mg / m 2 ( / day) and the specified dose of fludarabine (20 mg / m²) 2 / day~900mg / m 2This document describes a method for conditioning patients requiring T-cell therapy, including administering the patient approximately 500 mg / m² / day. One such dose regimen involves administering the patient approximately 500 mg / m² before administering a therapeutically effective dose of engineered T cells to the patient. 2 Cyclophosphamide per day, and approximately 60 mg / m² 2 The treatment of the patient includes administering fludarabine at a dose of 500 mg / m² daily for three days, including 4, 3, and 2 days prior to T cell administration. Another embodiment involves administering serum cyclophosphamide and fludarabine at a dose of 500 mg / m² daily during that period. 2 Dosage of cyclophosphamide based on body surface area and 30 mg / m² per day 2 The dosage of fludarabine is based on body surface area. Another embodiment involves cyclophosphamide on day -2 prior to T cell administration, and fludarabine on days -4, -3, and -2, at a rate of 900 mg / m² per day during that period. 2 Body surface area and cyclophosphamide dosage 25 mg / m² 2 The dosage of fludarabine is based on the body surface area. In another embodiment, conditioning involves cyclophosphamide and fludarabine on days -5, -4, and -3 prior to T cell administration, at a rate of 500 mg / m² per day during that period. 2 Dosage of cyclophosphamide based on body surface area and 30 mg / m² per day 2 The dosage of fludarabine is calculated based on body surface area. Other preconditioning regimens include 200-300 mg / m² per day for 3 days. 2 Cyclophosphamide by body surface area and 20-50 mg / m² per day 2 Contains fludarabine in a dose based on body surface area. In some embodiments, CAR T cell therapy is performed on day 0 with 2 × 10⁶ cells. 6 This involves a single IV infusion of 10 CAR T cells / kg. In some embodiments, at least about 10 4 Individual cells, at least about 10 5 Individual cells, at least about 10 6 Individual cells, at least about 10 7 Individual cells, at least about 10 8 Individual cells, at least about 10 9 pieces, or at least about 10 10A number of CAR T cells are administered. In another embodiment, the therapeutically effective dose of T cells is approximately 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of T cells is approximately 2 × 10⁻⁶ 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 4×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 6×10 6 Individual cells / kg, approximately 7×10 6 Individual cells / kg, approximately 8×10 6 Individual cells / kg, approximately 9×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 4×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 6×10 7 Individual cells / kg, approximately 7×10 7 Individual cells / kg, approximately 8×10 7 Individual cells / kg, or approximately 9 × 10⁻⁶ cells 7 The cell count is individual cells / kg. In some embodiments, the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cell therapy comprises anti-CD19 CAR T cells. In some embodiments, the CAR T cell therapy comprises axicabutagen silolucel or YESCARTA®. In some embodiments, the CAR T cell therapy comprises TECARTUS®-brexcabutagen silolucel or KYMRIAH® (tisagen lecleucel), etc., idekabutagen silolucel / bb2121.

[0202] In another embodiment, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, comprising administering a therapeutically effective dose of CD19 CAR-T therapy to the subject having received one to two, three, four, or five or more prior therapies. In one embodiment, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, comprising administering a therapeutically effective dose of CD19 CAR-T therapy to the subject having received one to two prior therapies. 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, the CD19 CAR-T therapy is used as a first-line treatment. In some embodiments, the CD19 CAR-T therapy is used as a second-line treatment.

[0203] In one embodiment, CD19 CAR-T therapy is any of the above-described CD19 CAR-T therapies. In one embodiment, CD19 CAR-T therapy includes axicaptagensilol-ucel therapy. In the embodiment, the cancer is treatment-resistant DLBCL non-specific type (ABC / GCB), HGBL with or without MYC and BCL2 and / or BCL6 rearrangements, DLBCL arising from FL, T-cell / histiocyte-rich large B-cell lymphoma, chronic inflammation-associated DLBCL, primary cutaneous DLBCL lower extremity type, and / or Epstein-Barr virus (EBV) positive DLBCL. In one embodiment, the subjects selected for axicapbutagensilol-ucel therapy include treatment-resistant DLBCL nonspecific type (ABC / GCB), HGBL with or without MYC and BCL2 and / or BCL6 rearrangement, DLBCL arising from FL, T-cell / histiocyte-rich large B-cell lymphoma, chronic inflammation-associated DLBCL, primary cutaneous DLBCL lower extremity type, and / or Epstein-Barr virus (EBV) positive DLBCL. In some embodiments, axicapbutagensilol-ucel therapy is used as a second-line treatment, where the first-line treatment is CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone. In some embodiments, axicaptagensilol-ucel therapy is used as a second-line treatment, where the first-line treatment is R-CHOP (CHOP plus rituximab).

[0204] In some embodiments, patients with relapsed or treatment-resistant disease after first-line chemoimmunotherapy are selected for second-line axicapbutagen silol-ucel treatment, where treatment-resistant disease is defined as the absence of complete remission to first-line treatment, and individuals intolerant to first-line treatment are excluded. The best response to first-line treatment is defined as disease progression (PD); the best response is defined as stable disease (SD) after at least four cycles of first-line therapy (e.g., four cycles of R-CHOP); the best response is defined as partial response (PR) after at least six cycles and residual lesions or disease progression as demonstrated by biopsy within 12 months of treatment; and / or disease relapse as demonstrated by biopsy within 12 months of first-line treatment following complete remission to first-line treatment. In some embodiments, patients selected for second-line axicapbutagen silol-ucel treatment receive fludarabine 30 mg / m² IV and cyclophosphamide 500 mg / m² on days -5, -4, and -3. 2 A conditioning therapy including IV is provided. In some embodiments, axicaptagen silolucel therapy is used as a second-line treatment. Combination therapy

[0205] Compositions comprising CAR-expressing immunoeffector cells disclosed herein may be administered in combination with any number of chemotherapeutic agents (before, after, and / or simultaneously with T cell administration). In some embodiments, antigen-binding molecules, transduced (or otherwise manipulated) cells (e.g., CARs), and chemotherapeutic agents are each administered in amounts effective to treat the disease or condition of interest. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carbocon, metredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotosine, fotemustine, ro Mustine, nimustine, ranimustine, antibiotics, for example, acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, Mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, pofilomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate;Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenaline, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatrexate; defofamine; demecoltin; diaziquan; erfol Mitin; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllic acid, 2-ethylhydrazide; procarbazine; polysaccharide K (PSK); razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeroda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomitine (DMFO); retinoic acid derivatives, e.g., Targretin® (bexarotene), Panretin® (allitretinoin);Examples include ONTAK™ (denileukin difutitox); esperamycin; capecitabine, and any pharmaceutically acceptable salts, acids, or derivatives of the above. In some embodiments, compositions comprising CAR-expressing immunoeffector cells disclosed herein may be administered in combination with anti-estrogens, such as tamoxifen, raloxifene, 4(5)-imidazole (an aromatase inhibitor), 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of the above. If necessary, but not limited to, combinations of chemotherapy drugs including CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, R-CHOP (CHOP plus rituximab), and G-CHOP (CHOP plus obinutuzumab) may also be administered.

[0206] In some embodiments, the chemotherapeutic agent is administered simultaneously or within one week after administration of the manipulated cells. In other embodiments, the chemotherapeutic agent is administered for 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the manipulated cells or nucleic acids. In some embodiments, the chemotherapeutic agent is administered at least one month before administration of the cells or nucleic acids. In some embodiments, the method further includes the administration of two or more chemotherapeutic agents.

[0207] Various additional therapeutic agents may be used in conjunction with the compositions described herein (before T-cell administration, after T-cell administration, and / or concurrently with T-cell administration). For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), semiprimab (Libtayo), pidilizumab (CureTech), and atezolizumab (Roche), as well as PD-L1 inhibitors, such as atezolizumab, durvalumab, and avelumab. In some embodiments, the combination therapy(s) may be selected from anti-IL-1 (e.g., anakinra), T-cell activating inhibitors (e.g., dasatinib), JAK inhibitors (e.g., filgotinib), anti-GM-CSF (e.g., renzilumab), anti-TNF (e.g., infliximab), Ang2 inhibitors (e.g., azilsartan), anti-angiogenic therapies (e.g., bevacizumab), and / or anti-IFNg (e.g., emaparmab-lzsg).

[0208] Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein (before T-cell administration, before and after T-cell administration, and / or concurrently with T-cell administration) include, but are not limited to, ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), and trastuzumab (HERCEPTIN®). Trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), katumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, macitinib, pazopani Bu, sunitinib, sorafenib, toceranib, restortinib, axitinib, cejiranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, restortinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, Examples of antithymocyte inhibitors include binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin difutitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidedib and bismodegib, CDK inhibitors such as CDK inhibitors (palbociclib), GM-CSF, CSF1, GM-CSFR, or CSF1R inhibitors, as well as antithymocyte globulin, rengilumab, and mabrilimumab.

[0209] In one embodiment, the GM-CSF inhibitor is selected from rengilumab; namilumab (AMG203); GSK3196165 / MOR103 / ochilimab (GSK / MorphoSys); KB002 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022 / demicirumab (Morphotek); or any of these biosimilars; E21R; and small molecules. In one embodiment, the CSF1 inhibitor is selected from RG7155, PD-0360324, MCS110 / lacnotuzumab, or any of these biosimilars; and small molecules. In one embodiment, the GM-CSFR inhibitor and CSF1R inhibitor are selected from maprilimumab (formerly CAM-3001; MedImmune, Inc.); kabilizumab (Five Prime Therapeutics); emactuzumab, also known as LY3022855 (IMC-CS4) (Eli Lilly), 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); any of these biosimilar versions; and small molecules.

[0210] In some embodiments, compositions comprising immunotherapy (e.g., engineered CAR T cells) are administered together with anti-inflammatory agents (before T cell administration, after T cell administration, and / or concurrently with T cell administration). Examples of anti-inflammatory agents include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) (including aspirin, ibuprofen, and naproxen), methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolates. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Examples of analgesics include acetaminophen, oxycodone, and tramadol (proporxiphen hydrochloride). Examples of glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers also include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0211] In some embodiments, the compositions described herein are administered together with cytokines (before, after, or concurrently with T cell administration). Examples of cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin; glycoprotein hormones, e.g., follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone. hormone (LH), hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin, placental lactogen, murelan inhibitor; mouse gonadotropin-related peptide; inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF), e.g., NGF-beta; platelet growth factor; transforming growth factor (TGF), e.g., TGF-α and TGF-β; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO, Epogen®, Procrit®); bone induction factor, inter These include interferons, e.g., interferon-alpha, beta, and gamma; colony-stimulating factors (CSFs), e.g., macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), e.g., IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15; tumor necrosis factors, e.g., TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligands (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, and biologically active equivalents of naturally occurring cytokines.

[0212] In some embodiments, the administration of cells and the administration of additional therapeutic agents are performed on the same day, or at intervals of 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or 30 minutes or less. In some embodiments, the administration of cells and the administration of additional therapeutic agents are performed between 0 hours or about 0 hours and 48 hours or about 48 hours, between 0 hours or about 0 hours and 36 hours or about 36 hours, between 0 hours or about 0 hours and 24 hours or about 24 hours, between 0 hours or about 0 hours and 12 hours or about 12 hours, between 0 hours or about 0 hours and 6 hours or about 6 hours, between 0 hours or about 0 hours and 2 hours or about 2 hours, or between 0 hours or about 0 hours and 1 hour or about 1 hour Between 0 hours or approximately 0 hours and 30 minutes or approximately 30 minutes, between 30 minutes or approximately 30 minutes and 48 hours or approximately 48 hours, between 30 minutes or approximately 30 minutes and 36 hours or approximately 36 hours, between 30 minutes or approximately 30 minutes and 24 hours or approximately 24 hours, between 30 minutes or approximately 30 minutes and 12 hours or approximately 12 hours, between 30 minutes or approximately 30 minutes and 6 hours or approximately 6 hours, between 30 minutes or approximately 30 minutes and 4 hours or approximately 4 hours, between 30 minutes or approximately 30 minutes and 2 hours or approximately 2 hours, Between 30 minutes or approximately 30 minutes and 1 hour or approximately 1 hour, between 1 hour or approximately 1 hour and 48 hours or approximately 48 hours, between 1 hour or approximately 1 hour and 36 hours or approximately 36 hours, between 1 hour or approximately 1 hour and 24 hours or approximately 24 hours, between 1 hour or approximately 1 hour and 12 hours or approximately 12 hours, between 1 hour or approximately 1 hour and 6 hours or approximately 6 hours, between 1 hour or approximately 1 hour and 4 hours or approximately 4 hours, between 1 hour or approximately 1 hour and 2 hours or approximately 2 hours, 2 hours Between approximately 2 hours and 48 hours, between approximately 2 hours and 36 hours, between approximately 2 hours and 24 hours, between approximately 2 hours and 12 hours, between approximately 2 hours and 6 hours, between approximately 2 hours and 4 hours, between approximately 4 hours and 48 hours, between approximately 4 hours and 36 hours,It will be executed between 4 hours or approximately 4 hours and 24 hours or approximately 24 hours, between 4 hours or approximately 4 hours and 12 hours or approximately 12 hours, between 4 hours or approximately 4 hours and 6 hours or approximately 6 hours, between 6 hours or approximately 6 hours and 48 hours or approximately 48 hours, between 6 hours or approximately 6 hours and 36 hours or approximately 36 hours, between 6 hours or approximately 6 hours and 24 hours or approximately 24 hours, between 6 hours or approximately 6 hours and 12 hours or approximately 12 hours, between 12 hours or approximately 12 hours and 48 hours or approximately 48 hours, between 12 hours or approximately 12 hours and 36 hours or approximately 36 hours, between 12 hours or approximately 12 hours and 24 hours or approximately 24 hours, between 24 hours or approximately 24 hours and 48 hours or approximately 48 hours, between 24 hours or approximately 24 hours and 36 hours or approximately 36 hours, or between 36 hours or approximately 36 hours and 48 hours or approximately 48 hours. In some embodiments, cells and additional therapeutic agents are administered simultaneously.

[0213] In some embodiments, the drug is administered in doses of 30 mg or approximately 30 mg to 5000 mg, for example, 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.

[0214] In some embodiments, the drug is administered in doses 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, or 50 mg / kg to 100 mg / kg. In some embodiments, the drug is administered in doses of 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, respectively. In some embodiments, the drug is administered in doses of at least 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, or higher.

[0215] In some embodiments, the drug(s) are administered by injection, such as intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, choroidal injection, anterior chamber injection, subconjunctival injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or delivery near the posterior sclera. In some embodiments, they are administered by parenteral administration, intrapulmonary administration, intranasal administration, or intralesional administration if local treatment is desired. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0216] In some embodiments, the treatment further includes a bridging therapy, which is a treatment between conditioning and the compositions disclosed herein, or a treatment administered after leukocyte apheresis and completed before initiating conditioning chemotherapy. In some embodiments, the bridging therapy includes 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 duverisib, idelalisib, venetoclax, pictilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), piraralisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselicib (GDC-0032). In some embodiments, the AKT inhibitor is perifosine or MK-2206. In one embodiment, the mTOR inhibitor is selected from everolimus, sirolimus, temsirolimus, and ridaflorimus. In some embodiments, the PI3K / mTOR dual 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), piraralisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselicib (GDC-0032).

[0217] In some embodiments, bridging therapy includes acalabrutinib, brentuximab vedotin, copanlisib hydrochloride, nelarabine, bellinostat, bendamustine hydrochloride, carmustine, bleomycin sulfate, bortezomib, zanubrutinib, carmustine, chlorambucil, copanlisib hydrochloride, denileukin difutitox, dexamethasone, doxorubicin hydrochloride, duvelisib, pralatrexate, obinutuzumab, ibritumomab tiuxetan, ibrutinib, idelalisib, recombinant interferon α-2b, romidepsin, lenalidomide, and mechloret. This includes methyl hydrochloride, methotrexate, mogamulizumab-kpc, plerixafor, nelarabine, obinutuzumab, deniloukin difutitox, pembrolizumab, plerixafor, polatuzumab vedotin-piiq, mogamulizumab-kpc, prednisone, rituximab, hyaluronidase, romidepsin, bortezomib, venetoclax, vinblastine sulfate, vorinostat, zanubrutinib, CHOP, COPP, CVP, EPOCH, R-EPOCH, HYPER-CVAD, ICE, R-ICE, R-CHOP, R-CVP, and combinations thereof.

[0218] In some embodiments, cellular immunotherapy is administered in conjunction with decompression therapy used to reduce systemic tumor tissue volume. In one embodiment, decompression therapy should be administered after leukocyte apheresis and before the administration of conditioning chemotherapy or cell infusion. Examples of decompression therapy are given below (Table 1).

[0219] [Table 1] Abbreviation: AUC, area under the curve a Other weight-loss treatment options may be used and will be discussed with the medical monitor. Supportive therapies such as hydration, antiemetics, mesna, growth factor support agents, and tumor lysis prevention may be used according to local standards. More than one cycle is permitted. b To enable tumor measurement, at least one target lesion is left outside the radiation field. monitoring

[0220] In some embodiments, the administration of immunotherapy (e.g., chimeric receptor T-cell immunotherapy) is performed at an accredited medical institution.

[0221] In some embodiments, the methods disclosed herein include monitoring the patient daily for at least seven days after infusion at an accredited medical facility for signs and symptoms of CRS and neurotoxicity and other adverse reactions to CAR T-cell therapy. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are instructed to remain near an accredited medical facility for at least four weeks after the injection. Prevention or management of adverse events

[0222] In some embodiments, the method includes the management of adverse events in any subject. The terms “adverse event,” “adverse reaction,” and “adverse action” are used interchangeably herein. In some embodiments, the adverse event is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, serious infections, cytopenia, and hypogammaglobulinemia.

[0223] In some embodiments, this disclosure provides methods for preventing the onset of adverse events or reducing the severity of adverse reactions based on levels of numerous biomarkers in the serum of subjects receiving immunotherapy. In some embodiments, cell therapy is administered with one or more agents that prevent, delay the onset of, reduce the symptoms of, or treat adverse events, including cytokine release syndrome and neurotoxicity. In one embodiment, the agents are described above. In other embodiments, the agents are described below. In some embodiments, the agents are administered before, after, or concurrently with cell administration by one of the methods and dosages described elsewhere in this specification. In one embodiment, the agents are administered to subjects who may be prone to disease but have not yet been diagnosed with the disease.

[0224] In some embodiments, immunotherapy (e.g., cell therapy) is administered before, during / concurrently with, and / or after the administration (e.g., steroids) or treatment (e.g., dose reduction) of one or more agents that treat or prevent (are prophylactic) one or more symptoms of an adverse event. The pharmacological and / or physiological effects may be prophylactic, i.e., the effect completely or partially prevents the disease or its symptoms. "Prophylactic effective dose" refers to the effective dose to achieve the desired prophylactic outcome in the required dose and duration. In one embodiment, the prophylactic effective dose is used in a subject prior to or at an early stage of the disease. In one embodiment, the prophylactic effective dose will be less than the therapeutic effective dose. In some embodiments, patients are selected for the management of adverse events based on the expression of one or more markers described herein. In one embodiment, the treatment or prevention of adverse events is administered to any patient who is, is, or has previously received cell therapy.

[0225] In some embodiments, signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0226] In some embodiments, patients are identified and selected based on one or more of the biomarkers described in this application. In some embodiments, patients are identified and selected simply by clinical symptoms (e.g., presence and grade of toxic symptoms).

[0227] In some embodiments, adverse events / reactions may be selected from one or more of the following (Table 2).

[0228] [Table 2] Cytokine release syndrome (CRS)

[0229] In some embodiments, the method includes preventing CRS in chimeric receptor therapy or reducing its severity. In some embodiments, the manipulated CAR T cells are inactivated after administration to the patient.

[0230] In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. Patients exhibiting Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid resuscitation, or hypoxia requiring oxygen supplementation) should be monitored with continuous telecardiogram monitoring and pulse oximetry. In some embodiments, patients exhibiting severe CRS should be considered for echocardiography to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered.

[0231] In some embodiments, the method includes monitoring the patient daily for at least 7 days after infusion at an accredited medical facility for signs and symptoms of CRS. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention at a hospital whenever signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as an indication for the first signs of CRS.

[0232] In some embodiments, the subject experiences Grade 3+ CRS. In some embodiments, these include fever, hypotension, tachycardia, hypoxia, chills, sinus tachycardia, malaise, headache, vomiting, acute kidney injury, myalgia, atrial fibrillation, diarrhea, dyspnea, decreased ejection fraction, pulmonary edema, atrial flutter, increased serum creatinine, capillary leak syndrome, decreased appetite, febrile neutropenia, malaise, metabolic acidosis, fever, nausea, headache, rash, rapid heartbeat, hypotension, and dyspnea. Neurotoxicity (NT)

[0233] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients undergoing Grade 2 or higher neurotoxicity should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

[0234] In some embodiments, the subject experiences Grade 3+ NT. In some embodiments, this includes brain damage, tremor, confusional state, aphasia, somnolence, agitation, memory impairment, dysarthria, hallucinations, altered mental state, ataxia, restlessness, seizures, delirium, attention deficit, apathy, decreased level of consciousness, disorientation, dyscalculia, unilateral hemiparesis, monoclonal spondylosis, and cerebral edema. Management of adverse events

[0235] In some embodiments, the method for managing adverse events includes monitoring the patient daily for at least 7 days after infusion at an accredited medical facility for signs and symptoms of neurotoxicity. In some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity and / or CRS, or for 4 weeks after infusion.

[0236] In some embodiments, the Disclosure provides two methods for managing adverse events in subjects receiving CAR T-cell therapy with steroids and anti-IL6 / anti-IL-6R antibodies. In one embodiment, the CAR T-cell therapy is anti-CD19 therapy, as described in the Examples. In one embodiment, the CAR T-cell therapy is known as ZUMA-1, which includes different adverse event management protocols for different cohorts. In one embodiment, the Disclosure presents that early steroid intervention in cohort 4 is associated with a lower rate of severe CRS and neurological events compared to those observed in cohorts 1+2. In one embodiment, the Disclosure presents that early use of steroids in cohort 4 resulted in a median cumulative cortisone equivalent dose being approximately 15% of that in cohorts 1+2, suggesting that early steroid use may allow for a reduction in total steroid exposure. Accordingly, in one embodiment, the Disclosure provides a method for managing adverse events in which corticosteroid therapy is initiated for the management of all cases of grade 1 CRS if no improvement is seen after 3 days for all grade 1 or higher neurological events. In one embodiment, the disclosure states that if no improvement is observed after 3 days for all Grade ≥ 2 neurological events, tocilizumab is initiated for the management of all cases of Grade 1 CRS. In one embodiment, the disclosure provides a method for reducing total steroid exposure in patients receiving adverse event management after CAR T cell therapy, the method comprising initiating corticosteroid therapy for the management of all cases of Grade 1 CRS if no improvement is observed after 3 days for all Grade ≥ 1 neurological events, and / or initiating tocilizumab for all cases of Grade 1 CRS if no improvement is observed after 3 days for all Grade ≥ 2 neurological events. In one embodiment, corticosteroids and tocilizumab are administered in regimens selected from those exemplified in protocols A-C. In one embodiment, the disclosure provides that early steroid use is not associated with an increased risk of severe infection, decreased CAR T cell proliferation, or decreased tumor response.

[0237] In one embodiment, this disclosure supports the safety of levetiracetam prophylaxis in CAR T-cell cancer therapy. In one embodiment, the cancer is NHL. In another embodiment, the cancer is R / R LBCL, and the patient is administered axicaptagen silolucel. Thus, in one embodiment, this disclosure provides a method for managing adverse events in a patient treated with CAR T cells, comprising administering a prophylactic dose of an anticonvulsant to the patient. In some embodiments, the patient is administered levetiracetam (e.g., 750 mg orally or intravenously, twice daily) starting on day 0 of CAR T-cell therapy (post-pre-treatment), and if a neurological event occurs after discontinuation of prophylactic levetiracetam, or upon development of grade ≥ 2 neurotoxicity. In one embodiment, if the patient does not experience any grade ≥ 2 neurotoxicity, levetiracetam is reduced and discontinued as clinically indicated. In one embodiment, levetiracetam prophylaxis is combined with any other adverse event management protocol.

[0238] In one embodiment, the disclosure shows that CAR T cell levels in patients subject to the Cohort 4 adverse event management protocol were equivalent to those in Cohort 1+2. In one embodiment, the disclosure shows that levels of major inflammatory cytokines associated with CAR-related inflammatory events (e.g., IFNγ, IL-2, and GM-CSF) were lower in Cohort 4 than in Cohort 1+2. Accordingly, the disclosure provides a method for reducing CAR T cell therapy-related inflammatory events without affecting CAR T cell levels, comprising administering the Cohort 4 adverse event management protocol to the patient. The disclosure also provides a method for reducing cytokine production by immune cells after CAR T cell therapy, comprising administering the Cohort 4 adverse event management protocol to the patient. In one embodiment, this effect is achieved without affecting CAR T cell proliferation and response rates. In one embodiment, the patient has R / R LBCL. In one embodiment, the CAR T cell therapy is anti-CD19 CAR T cell therapy. In one embodiment, CAR T cell therapy includes axicaptagensilolucel.

[0239] In one embodiment, the disclosure shows that early or prophylactic use of tocilizumab after axicaptagen silol for adverse event management reduced grade 3 or higher cytokine release syndrome but increased grade 3 or higher neurological events. Accordingly, the disclosure provides a method for adverse event management in CAR T-cell therapy. In one embodiment, the patient is administered levetiracetam starting on day 0 (750 mg twice daily, orally or intravenously). Upon development of a grade 2 or higher neurological event, the dose of levetiracetam is increased to 1000 mg twice daily. If the patient does not exhibit any grade 2 or higher neurological events, levetiracetam is tapered and discontinued as clinically necessary. On day 2, the patient is also administered tocilizumab (8 mg / kg IV over 1 hour [not exceeding 800 mg]). Further tocilizumab (±corticosteroids) may be recommended in patients with comorbidities or in elderly patients upon the onset of Grade 2 CRS, or otherwise in cases of Grade 3 or higher CRS. Tocilizumab is initiated in patients presenting with Grade 2 or higher neurological events, and corticosteroids are added in patients with comorbidities or in elderly patients, or in cases of Grade 3 or higher neurological events that worsen despite tocilizumab use.

[0240] In one embodiment, the disclosure shows that prophylactic use of steroids reduces the rate of severe CRS and NE to a level comparable to early steroid use after axicaptagen silol-ucel administration. Accordingly, the disclosure provides a method for managing adverse events in CAR T-cell therapy, wherein the patient is orally administered 10 mg of dexamethasone on day 0 (before axicaptagen silol-ucel infusion), day 1, and day 2. Steroids are also administered in the event of grade 1 NE and for grade 1 CRS if no improvement is observed after 3 days of supportive care. Tocilizumab is also administered for grade 1 or higher CRS if no improvement is observed after 24 hours of supportive care.

[0241] In one embodiment, the disclosure presents that treatment-related CRS and / or NE in treated patients can be prevented or reduced by managing adverse events of CAR T cell therapy with an antibody that neutralizes and / or depletes GM-CSF. In one embodiment, the antibody is lenzirumab.

[0242] In one embodiment, a method for preventing and / or managing adverse events includes the administration of a “prophylactic effective dose” of tocilizumab, corticosteroid therapy, and / or anticonvulsants for toxic prophylaxis. In some embodiments, the method includes the administration of inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, renzilumab, maprilumab, cytokines, and / or anti-inflammatory agents.

[0243] In some embodiments, adverse events are controlled by administering a drug / multiple drugs that are antagonists or inhibitors of IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the drug is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the drug is or comprises the anti-IL-6R antibody tocilizumab (atlizumab) or sarilumab. In some embodiments, the drug is the anti-IL-6R antibody described in U.S. Patent No. 8,562,991. In some examples, IL-6 targeting agents are anti-IL-6 antibodies, such as siltuximab, elcirimomab, ALD518 / BMS-945429, sirukmab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), and combinations thereof. In some embodiments, the agent may neutralize IL-6 activity by inhibiting ligand-receptor interaction. In some embodiments, the IL-6 / IL-6R antagonist or inhibitor is IL-6 mutein, for example, as described in U.S. Patent No. 5591827. In some embodiments, the agent that is an IL-6 / IL-6R antagonist or inhibitor is a small molecule, protein or peptide, or nucleic acid.

[0244] In some embodiments, other agents that may be used to manage adverse reactions and their symptoms include cytokine receptors or cytokine antagonists or inhibitors. In some embodiments, the cytokine or receptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R / CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFα, TNFR1, for example, TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), IL-1, and IL-1Rα / IL-1β. In some embodiments, the drug includes siltuximab, sarilumab, olokizumab (CDP6038), elcilimomab, ALD518 / BMS-945429, silkumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the drug is a cytokine antagonist or inhibitor, such as transforming growth factor β (TGFβ), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNFα, IL-1, interferon γ (IFN-γ), or monocyte chemotactic protein-1 (MCP-1). In some embodiments, the drug targets cytokine receptors (e.g., inhibits or is an antagonist thereof), such as the TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP), or IL-10 receptor (IL-10R), and combinations thereof.In some embodiments, the drug is administered before, after, or concurrently with the administration of cells by one of the methods and dosages described elsewhere in this specification.

[0245] In some embodiments, the drug is administered in doses of 1 mg / kg to 10 mg / kg or about 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg or about 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg or about 2 mg / kg to 6 mg / kg, 2 mg / kg to 4 mg / kg or about 2 mg / kg to 4 mg / kg, or 6 mg / kg to 8 mg / kg or about 6 mg / kg to 8 mg / kg (including both ends of the range), or the drug is administered in doses of at least 2 mg / kg or at least about 2 mg / kg or about 2 mg / kg, at least 4 mg / kg or at least about 4 mg / kg or about 4 mg / kg, at least 6 mg / kg or at least about 6 mg / kg or about 6 mg / kg, or at least 8 mg / kg or at least about 8 mg / kg or about 8 mg / kg. In some embodiments, the drug is administered in doses of approximately 1 mg / kg to 12 mg / kg, for example, 10 mg / kg or approximately 10 mg / kg. In some embodiments, the drug is administered by intravenous infusion. In one embodiment, the drug is tocilizumab. In some embodiments, the drug, for example, tocilizumab in particular, is administered before, after, or concurrently with cell administration by one of the methods and doses described elsewhere in this specification.

[0246] In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. If CRS is observed or suspected, it may be managed according to the recommendations of Protocol A, and this may be used in combination with other treatments of the Disclosure, including neutralization or reduction of the CSF / CSFR1 system. Patients exhibiting Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid resuscitation, or hypoxia requiring oxygen supplementation) should be monitored with continuous telecardiogram monitoring and pulse oximetry. In some embodiments, for patients exhibiting severe CRS, consider performing echocardiography to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered. In some embodiments, a biosimilar or equivalent of tocilizumab may be used instead of tocilizumab in the methods disclosed herein. In other embodiments, another anti-IL6R may be used instead of tocilizumab.

[0247] In some embodiments, adverse events are managed according to the following protocol (Protocol A / Table 3).

[0248] [Table 3] (a) The procedure will be carried out 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. (b) Refer to Protocol B for the management of neurotoxicity. (c) For further details, see ACEMTRA® (tocilizumab) prescribing information, https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). The initial US approval was indicated in 2010. (d) Alternative therapies include, but are not limited to, anakinra, siltuximab, ruxolitinib, cyclophosphamide, IVIG, and ATG. Neurotoxicity

[0249] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients undergoing Grade 2 or higher neurotoxicity should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. Consider non-sedating antiseizure agents (e.g., levetiracetam) for seizure prevention in any Grade 2 or higher neurotoxicity. The following treatments may be used in combination with other treatments of the Disclosure, such as neutralization or reduction of the CSF / CSFR1 axis.

[0250] In some embodiments, the NE is managed according to the following protocol (Protocol B / Table 4).

[0251] [Table 4] a Severity based on Common Terminology Criteria for Adverse Events. b Alternative therapies include, but are not limited to, anakinra, siltuximab, ruxolitinib, cyclophosphamide, IVIG, and ATG. Methylprednisolone can be used as an equivalent substitute for dexamethasone. Further safety management methods using corticosteroids

[0252] The administration of corticosteroids and / or tocilizumab in Grade 1 can be considered prophylactic. Supportive care may be provided in all protocols for all CRS and NE severity grades.

[0253] In one embodiment of a protocol for managing adverse events associated with CRS, tocilizumab, and / or corticosteroids, the following administration is performed: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only in cases with comorbidities or advanced age); and / or corticosteroids (only in cases with comorbidities or advanced age); Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids. In another embodiment of the protocol for managing adverse events associated with CRS, tocilizumab and / or corticosteroids are administered as follows: Grade 1 CRS: tocilizumab (if no improvement after 3 days); and / or corticosteroids (if no improvement after 3 days); Grade 2 CRS: tocilizumab; and / or corticosteroids; Grade 3 CRS: tocilizumab; and / or corticosteroids; Grade 4 CRS: tocilizumab; and / or corticosteroids, high dose.

[0254] In one embodiment of a protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following is administered: Grade 1 NE: without tocilizumab; without corticosteroids;

[0255] Grade 2 NE: No tocilizumab; no corticosteroids; Grade 3 NE: Tocilizumab; and / or corticosteroids (standard dose, only if there is no improvement with tocilizumab); Grade 4 NE: Tocilizumab; and / or corticosteroids.

[0256] In another embodiment of the protocol for managing adverse events associated with NE, tocilizumab, and / or corticosteroids, the following is administered: Grade 1 NE: no tocilizumab; and / or corticosteroid; Grade 2 NE: tocilizumab; and / or corticosteroid; Grade 3 NE: tocilizumab; and / or corticosteroid, high dose; Grade 4 NE: tocilizumab; and / or corticosteroid, high dose.

[0257] In one embodiment, corticosteroid therapy is initiated at grade 2 or higher CRS, and tocilizumab is initiated at grade 2 or higher CRS. In another embodiment, corticosteroid therapy is initiated at grade 1 or higher CRS, and tocilizumab is initiated at grade 1 or higher CRS. In yet another embodiment, corticosteroid therapy is initiated at grade 3 or higher NE, and tocilizumab is initiated at grade 3 or higher CRS. In yet another embodiment, corticosteroid therapy is initiated at grade 1 or higher CRS, and tocilizumab is initiated at grade 2 or higher CRS. In some embodiments, prophylactic use of tocilizumab administered on day 2 may reduce the proportion of grade 3 or higher CRS.

[0258] In one embodiment, the protocol for treating adverse events includes Protocol C as follows (Table 5).

[0259] [Table 5] a A treatment method that is gradually reduced at the discretion of the clinical trial physician as symptoms improve; b Do not exceed 800 mg; AE indicates adverse events, CRS, or cytokine release syndrome; IV indicates intravenous administration, N / A indicates not applicable, and NE indicates neurological events.

[0260] Any corticosteroid may be suitable for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, these two are administered in combination. In some embodiments, the glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include alclomethasone, alginate, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasol, crocoltron (e.g., crocoltron pivalate), Cloprednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortibazole, deflazacol, desonide, desoxymethasone, dexamethasone (e.g., dexamethasone phosphate 21, dexamethasone acetate, dexamethasone sodium phosphate), diflorazone (e.g., diflorazone diacetate), diflucortalolone, difluprednate, enoxolone, flurazacol, fluclonide, fludrocortisone Flumethasone (e.g., fludrocortisone acetate), flumetasone (e.g., flumetasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, flucortin, flutrolone, fluorometholone (e.g., fluorometholone acetate), fluperolon (e.g., fluperolon acetate), flupredniden, fluprednisolone, flulandrenolide, fluticasone (e.g., fluticasone propionate), formocortal, Halcinonide, halobetasol, halomethasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone acetate, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemysuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate,Hydrocortisone valerate, roteprednol etabonate, mazipredone, medrisone, meprednisone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), parametasone (e.g., parametasone acetate), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone f Examples include, but are not limited to, arnesylates, prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulfobenzoic acid, prednisolone stearate, prednisolone tebutate, prednisolone tetrahydrophthalate), prednisone, prednival, prednylidene, rimexolone, thixocortol, and triamcinolone (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are described in detail, for example, in Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980) and Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) and any other editions, which are incorporated herein by reference. In some embodiments, the glucocorticoid is selected from cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a mineralocorticoid. Any other steroid may be used in the manner provided herein.

[0261] One or more corticosteroids may be administered in any dose and frequency that can be adapted to the severity / grade of adverse events (e.g., CRS and NE). The table above provides examples of administration regimens for managing CRS and NE, respectively. In another embodiment, corticosteroid administration includes oral or intravenous administration of 10 mg of dexamethasone 1 to 4 times per day. Another embodiment, sometimes referred to as “high-dose” corticosteroids, includes intravenous administration of methylprednisone 1 g / day alone or in combination with dexamethasone. In some embodiments, one or more corticosteroids are administered at a dose of 1 to 2 mg / kg per day.

[0262] Corticosteroids may be administered in any amount effective in improving one or more symptoms associated with adverse events such as CRS or neurotoxicity. Corticosteroids, such as glucocorticoids, can be administered to a 70kg adult human subject in amounts of 0.1-100mg or approximately 0.1-100mg, 0.1-80mg, 0.1-60mg, 0.1-40mg, 0.1-30mg, 0.1-20mg, 0.1-15mg, 0.1-10mg, 0.1-5mg, 0.2-40mg, 0.2-30mg, 0.2-20mg, 0.2-15mg, 0.2-10mg, 0.2-5mg, 0.4-40mg, 0.4-30mg, 0.4-20mg, 0.4-15mg, 0.4-10mg, 0.4-5mg, 0.4-4mg, 1-20mg, 1-15mg, or 1-10mg per dose. Typically, corticosteroids such as glucocorticoids are administered to the average adult human subject in doses of approximately 0.4 to 20 mg, for example, approximately 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0263] In some embodiments, the corticosteroid is typically administered to an average adult subject weighing approximately 70-75 kg in doses such as, for example, 0.001 or approximately 0.001 mg / kg (of the subject), 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.035 mg / kg, 0.04 mg / kg, 0.045 mg / kg, 0.05 mg / kg, 0.055 mg / kg, 0.06 mg / kg, 0.065 mg / kg, 0.07 mg / kg, 0.075 mg / kg. g, 0.08mg / kg, 0.085mg / kg, 0.09mg / kg, 0.095mg / kg, 0.1mg / kg, 0.15mg / kg, 0.2mg / kg, 0.25mg / kg, 0.30mg / kg, 0.35mg / kg, 0.40mg / kg, 0.45mg / kg, 0.50mg / kg, 0.55mg / kg, 0.60mg / kg, 0.65mg / It can be administered in doses of kg, 0.70 mg / kg, 0.75 mg / kg, 0.80 mg / kg, 0.85 mg / kg, 0.90 mg / kg, 0.95 mg / kg, 1 mg / kg, 1.05 mg / kg, 1.1 mg / kg, 1.15 mg / kg, 1.20 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.35 mg / kg, or 1.4 mg / kg.

[0264] Generally, the dose of corticosteroid administered depends on the specific corticosteroid, as there are differences in potency between different corticosteroids. Typically, it is understood that because drugs have different potencies, the doses required to achieve equivalent effects may differ. Equivalent doses in terms of potency for various glucocorticoids and routes of administration are well known. Information on equivalent steroid doses (non-chronotherapy) can be found in the British National Formulary (BNF) 37, March 1999.

[0265] In some embodiments, adverse events are managed by the following protocol: The patient is administered levetiracetam (750 mg orally or intravenously twice daily) starting on day 0 of T-cell therapy administration; if a grade 2 or higher neurological event occurs, the levetiracetam dose is increased to 1000 mg twice daily; if the patient does not exhibit any grade 2 or higher neurological events, levetiracetam is gradually tapered and discontinued as clinically necessary; on day 2, the patient is also given tocilizumab (8 mg / kg IV is administered over 1 hour (not exceeding 800 mg); further tocilizumab (± corticosteroids) may be recommended in patients with comorbidities or elderly patients upon the onset of grade 2 CRS, or otherwise in cases of grade 3 or higher CRS; tocilizumab is initiated in patients presenting with grade 2 or higher neurological events, and corticosteroids are added in patients with comorbidities or elderly patients, or in cases of grade 3 or higher neurological events that worsen despite the use of tocilizumab. In some embodiments, levetiracetam is administered prophylactically, and in the event of grade 2 or higher neurotoxicity occurring if neurological events occur after discontinuation of prophylactic levetiracetam, and / or if the patient does not present with any grade 2 or higher neurotoxicity, levetiracetam is tapered and discontinued.

[0266] In some embodiments, adverse events are managed by the following protocol: the patient is orally administered 10 mg of dexamethasone on day 0 (before T-cell therapy infusion), day 1, and day 2; steroids are also administered in the event of a grade 1 NE and for grade 1 CRS if no improvement is observed after 3 days of supportive care; tocilizumab is also administered for grade 1 or higher CRS if no improvement is observed after 24 hours of supportive care. Secondary malignant tumors

[0267] In some embodiments, patients treated with CAR T cells (e.g., CD19-targeted) or other genetically modified autologous T cell immunotherapy may develop secondary malignancies. In certain embodiments, patients treated with CAR T cells (e.g., CD19-targeted) or other genetically modified allogeneic T cell immunotherapy may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies.

[0268] The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, dictionaries, documents, manuscripts, genome database sequences, and scientific literature cited herein are incorporated herein by reference. All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference. However, the reference of references herein should not be construed as an acknowledgment that such references are prior art to this disclosure. If any definition or term provided in an incorporated reference differs from the terms and definitions provided herein, these terms and definitions shall prevail.

[0269] The following embodiments are intended to illustrate various aspects of this application. Therefore, any particular embodiment considered should not be construed as a limitation on the scope of this application. For example, the following embodiments involve T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), but those skilled in the art will understand that the methods described herein may be applied to immune cells transduced with any CAR. The methods are also applicable to other immunotherapies. Those skilled in the art will understand that various equivalents, modifications, and alterations can be made without departing from the scope of this application, and that such equivalent embodiments are included herein.

[0270] The disclosure provided in this application may be used in various ways in addition to the methods described above, or in combination with the methods described above. The following is a summary of exemplary methods that may be derived from the disclosure provided in this application. [Examples]

[0271] Example 1 High B-cell signatures were associated with improved EFS and a higher probability of a persistent response after axi-cel. To identify novel prognostic markers in LBCL, gene expression analysis was performed on pre-treated tumor biopsies (collected either at initial diagnosis or before lymphocyte depletion chemotherapy), and predetermined gene expression signatures were evaluated using the NanoString PanCancer IO360™ panel (Table 6). In patients treated with second-line axi-cel therapy, the B-cell signature (IO360™) was associated with a higher likelihood of ongoing response (for both post-response and non-response progression); and improved EFS and duration of response (DOR; P<.05; patients with high signature values ​​[>median] versus patients with low values ​​[≤median] (P<.05). Pre-identified B-cell lineage signatures included BLK, CD19, MS4A1, TNFRSF17, FCRL2, FAM30A, PNOC, SPIB, and TCL1A. Of these genes, the expression of CD19, MS4A1, and TNFRSF17 was significantly elevated (description P<.05) in patients with ongoing response, with increases of 22%, 40%, and 69%, respectively. Conversely, hypoxia, nitric oxide synthase 2 (NOS2), and NKCD56dim were associated with Expression of the NanoString signature (composed of IL21R, KIR2DL3, KIR3DL1, and KIR3DL2) was associated with shorter EFS and / or DOR (P<.05).

[0272] In the SOC arm, B cell signatures were not associated with efficacy, and there were few NanoString signatures associated with the efficacy endpoint. Macrophage, myeloid, antigen-presenting mechanism (APM), natural killer (NK), or CD8 T cell immunogene expression signatures were slightly associated with any of the ongoing response, EFS, and / or DOR (no signature was consistently associated with all three efficacy metrics), suggesting that enrichment of selected tumor immunoinfiltrates may be a supporting factor for the SOC response. Nevertheless, axi-cel EFS was improved for SOC in all subgroups, including those with high APM (>median).

[0273] [Table 6-1] [Table 6-2] [Table 6-3]

[0274] In the table above, genes negatively associated with the clusters in which they are found are shown in bold. All other genes showed a positive association with the clusters. APM, antigen processing mechanism; IFN, interferon, JAK, Janus kinase; MAGE, melanoma antigen gene, MHC, major histocompatibility complex; MMR, mismatch repair; MSI, microsatellite instability; NK, natural killer; STAT, signaling transcription factor, Th1, T helper type 1; TIS, tumor inflammation signature; Treg, regulatory T cell. Clustering revealed different gene expression signatures consistent with different TME immune states.

[0275] Based on unsupervised clustering analysis of NanoString IO360™ results, four major gene expression signature clusters underlying different tumor microenvironment (TME) immune statuses were identified (Table 6). The signs within each cluster were combined to create an index for further analysis. The first cluster, referred to herein as B-cell lineage and proliferation index (BPI), included B-cell-like signatures, proliferation, APM loss, and glycolytic activity. Signals from BPI showed the highest hierarchical separation from the other three major clusters, suggesting a relatively simple TME with abundant and highly proliferative cancer cells and lower immune cell infiltration compared to the other clusters. The second cluster, called stroma and immunosuppression index (SII), characterized a set of genes including stromal cells, myeloid cells, and endothelial cells, NOS2, transforming growth factor β (TGF-β), B7-H3, arginase 1 (ARG1), and hypoxia. In this cluster, hypoxia and the NOS2 signature (IO360) were negatively associated with EFS and / or DOR after axi-cel treatment. A third cluster was enriched for NK cell, macrophage, and antigen-presenting cell signatures. A fourth cluster consisted mainly of T cell infiltration features. The third and fourth clusters showed relatively close hierarchical correlations and may together represent more complex, immunoinfiltrated tumors. SII and BPI (TME status) related to EFS within the axi-cel arm

[0276] Root mean square exponents were generated from each of the four clusters of NanoString signatures (see Methods). BPI (cluster 1) and SII (cluster 2) were positively and negatively associated with EFS in the axi-cel arm, respectively (description P < 0.05). None of the four clusters were associated with the cells of origin. Notably, BPI was positively associated with high-grade B-cell lymphoma (HGBL) and double-hit / triple-hit disease. The median EFS for the HGBL subgroup after axi-cel treatment was 21.5 months (95% CI, 3.7 - not quantifiable; unstratified HR [axi-cel over SOC] = 0.318). EFS for HGBL subtypes was not significantly different from that of non-HGBL subtypes (DLBCL + others), but a favorable directional EFS was observed between HGBL and non-HGBL subtypes in the axi-cel arm (unstratified HR [HGBL over non-HGBL] = 0.692; 95% CI, 0.384–1.245), suggesting that HGBL patients may benefit more from axi-cel compared to other patients. Conversely, the SOC arm showed a non-significant directional association in the opposite direction (unstratified HR [HGBL over 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 these four clusters were significantly associated with outcomes in the SOC arm, indicating different effects of TME on outcomes with SOC and CAR T-cell therapy. CD19 expression levels had different effects on TME and treatment-based efficacy.

[0277] CD19 protein levels (H score) correlated with CD19 gene expression and B cell signature. Consistent with the role of B cell signature in axi-cel mediated efficacy, CD19 gene expression and protein levels also correlated with axi-cel EFS. Axi-cel EFS was better in patients with high (>median) CD19 gene and protein expression levels compared to patients with lower expression (≤median). Notably, axi-cel remained superior to SOC across CD19 gene expression subgroups. Patients considered CD19-negative by immunohistochemistry (H score <5) still showed a substantial response to axi-cel, with an objective response rate (ORR) of 85% versus 67% in the SOC arm.

[0278] Patients with lower CD19 protein expression (H score ≤ median) had more complex immunoinfiltrative TMEs enriched with multiple immunosuppressive features, including regulatory T cells, T cell exhaustion, ARG1, indoleamine 2, 3-dioxygenase 1 (IDO1), B7-H3, CTLA4, and macrophage and myeloid gene expression signatures. The lowest EFS after axi-cel treatment was observed in patients with tumors having both low CD19 protein expression and high SII, suggesting that both TME immunosuppression and target expression play a role in resistance to CAR T cell therapy. Conversely, in patients with higher CD19 protein expression (> median), persistent lack of response was associated with glycolytic activity. Axi-cel products enriched with CCR7+CD45RA+ T cells can overcome low antigen (CD19) expression.

[0279] Naive-like T cells or stem memory phenotype (T SCMWe further investigated whether axi-cel products enriched with CCR7+CD45RA+ T cells (considered to be CCR7+CD45RA+ T cells) could overcome the adverse effects of undesirable TME. Indeed, patients with relatively low CD19 expression showed improved EFS when a higher frequency of CCR7+CD45RA+ T cells was present in the product. Similar results were obtained with respect to the total number of injected CCR7+CD45RA+ T cells. Patients with relatively high SII also showed improved EFS with a higher frequency of CCR7+CD45RA+ T cells in the product, although the difference was not statistically significant by descriptive statistics.

[0280] ZUMA-7 is the largest clinical dataset available in the context of CAR T-cell therapy for second-line LBCL. Here, we investigated the ZUMA-7 dataset to identify novel tumor biomarkers associated with responses (EFS, DOR, ongoing response, CR, objective response) to CAR T-cell therapy (axi-cel) or SOC (HDT-ASCT). We determined that outcomes for axi-cel or SOC were differentially influenced by the composition of the TME in the second-line setting, providing insights into the putative mechanisms driving responsiveness to these therapies. For example, B-cell scores and tumor gene expression signatures containing clusters enriched with stromal and immunosuppressive features (SII) were positively and negatively associated with CAR-T-cell therapy outcomes, respectively. In particular, within the B-cell score, CD19 gene and protein expression were also positively associated with cell therapy outcomes.

[0281] The analyses described herein identified major clusters of gene expression signatures. SII (cluster 2 in Table 6), negatively associated with clinical outcomes, likely reflect myelosuppression and immunosuppressive TMEs, including TGF-β activating stromal genes. Within this immune context, CAR T cells may not be adequately transported to malignant cells or maintain their functional state. In contrast, unexpectedly, BPI (cluster 1 in Table 6) was positively associated with HGBL / double-hit / triple-hit disease and favorable clinical outcomes. This high-risk subgroup was more likely to have a higher BPI index and exhibited a more homogeneous malignant B-cell population with less diverse immune infiltration.

[0282] Another finding of this study was the clear distinction between axi-cel biomarkers and SOC biomarkers associated with outcomes. Interestingly, pre-identified B-cell lineage signatures and CD19 expression were positively associated with outcomes after axi-cel treatment, while other TME immunological features, including APM and DC, were positively associated with outcomes after SOC. This suggests a mechanistic difference between direct antigen binding of CD19 by CAR under axi-cel and coselection of endogenous immunity against tumor epitopes under SOC. Notably, outcomes with axi-cel were improved compared to outcomes with SOC for all presented biomarker subgroups.

[0283] In summary, these findings revealed novel gene expression signatures that have potential predictive value for axi-cel response. These findings also help guide therapeutic decisions, including whether patients would benefit from cell therapy as a second-line treatment over standard care. The four clusters of TME signatures described herein, as well as the association between clusters 1 (BPI) and 2 (SII) and CAR T-cell therapy outcomes, have not been previously reported.

[0284] Here, a strong association is shown between cluster 1 (B cell proliferation) and HGBL. B cell proliferation and stromal clusters have also been reported to be associated with best and worst outcomes, respectively. These findings may have important implications for the transition of CAR T cell therapy and other treatments to previous lines of treatment, as predictive values ​​for responses to these TME signatures may exist.

[0285] Previously ununderstood correlations between baseline CD19 expression and TME immune infiltration and axi-cel outcomes are reported herein. Gene expression profiling by IHC or NanoString revealed associations between CD19 expression and outcomes. Axi-cel showed improved EFS compared to SOC regardless of CD19 protein expression protein / H score. Nevertheless, the ZUMA-7 axi-cel arm highlighted that patients with lower CD19 protein expression showed more complex immune infiltration TME, and that the relatively short EFS of axi-cel in patients with relatively low CD19 protein expression (H score ≤ median) may depend not only on suboptimal target expression but also on the characteristics of the concurrently confounding immune status. Indeed, the association between axi-cel EFS and low CD19 H score associated with cluster 2 (SII), and the association between CD19 H score and EFS, appeared to be limited to patients with high SII indices.

[0286] Patients with reduced B-cell signatures and a less favorable tumor immunomicroenvironment exhibited worse clinical prospects, but the crucial question is whether viable product characteristics can help overcome such undesirable features. As presented herein, CAR T-cell products enriched with the CCR7+CD45RA+ T-cell phenotype may improve outcomes in patients with lower CD19 protein expression and higher immunosuppressive characteristics.

[0287] Knowledge of the immune status is essential for understanding the mechanism of action and potential long-term responses to CAR-T cell therapy. In addition to SPD, MTV, LDH, and target (CD19) expression, measurement of tumor immune status using Immunoscore, IS21, B cells, and stromal and immunosuppressive gene signatures has emerged as important and interrelated determinants of persistent response to axi-cel intervention. Previous intervention with CAR-T cell therapy was supported by a more conductive immune status for increased axi-cel activity, which may contribute to the superior efficacy of axi-cel compared to SOC in second-line LBCL across common prognostic subgroups. Furthermore, patients with HGBL / double-hit / triple-hit disease, features associated with worse outcomes to conventional chemoimmunotherapy, substantially benefited from axi-cel treatment. The enrichment of BPIs in HGBL / double-hit / triple-hit tumors, and therefore the higher prevalence of proliferative B cells, CD19 expression, and relatively low immunosuppressive cells, may highlight the sensitivity of this high-risk population to CD19-targeted CAR T-cell therapy compared to SOCs. Read patient samples and efficacy

[0288] Evaluable samples from patients in the safety analysis sets of ZUMA-7 (N=170) and ZUMA-1 Cohort 1+2 (N=101) were analyzed. The ZUMA-7 safety analysis set was defined as randomized patients who received at least one dose of axi-cel or SOC. The ZUMA-1 safety analysis set was defined as all patients treated with any dose of axi-cel. The studies were approved by the institutional review boards of each study site and conducted in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines.

[0289] The ZUMA-7 efficacy endpoints (ORR, best response, EFS, DOR, and ongoing response) utilized the primary analysis data cutoff date. EFS was defined as the time from randomization to the earliest date of disease progression according to the Lugano classification, initiation of new lymphoma treatment, or death from any cause. Ongoing response was defined as patients who had an ongoing response (CR or partial response (PR)) by 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. As previously reported, patients who achieved stable or progressive disease as the best response were included in the no-response category. To contextualize the selected findings, data from patients with evaluable samples from the ZUMA-1 central cohort 1+2 were included with a minimum follow-up of 60 months. Analysis of gene expression signs

[0290] The retrieval of tumor biopsies and the processing of formalin-fixed paraffin-embedded biopsy specimens were similar for ZUMA-1 and ZUMA-7. Gene expression and molecular subgroup analysis were performed using the NanoString PanCancer IO360® panel and lymphoma subtyping tests. Predefined gene expression signatures from NanoString (proprietary algorithm) were analyzed for their association with clustering and efficacy readouts. Unsupervised clustering of gene expression signatures was performed in TIBCO Spotfire using a computed hierarchical clustering method (unweighted paired group method with arithmetic mean [UPGMA]; distance measurement - Euclidean, ordered weighted mean, empty value substitution method - constant values, no substitution with 0, and no normalization). Using Veracyte's proprietary algorithm, IS21, a predetermined gene expression signature for T cell infiltration and function, was calculated from gene expression values ​​in the PanCancer IO360® panel. Based on the PanCancer IO360™ panel, we investigated cell subtypes within TMEs and their association with clinical outcomes. Analysis of CD19 expression levels

[0291] CD19 protein expression levels were measured by IHC using an assay validated in NeoGenomics. Product attribute analysis

[0292] Product T cell phenotypes and other product attributes were evaluated as previously described. Further product characterization of co-stimulatory markers (CD27, CD28) and activation and exhaustion markers (programmed cell death protein 1, T cell immunoglobulin and mucin domain-containing protein 3, lymphocyte activation gene 3) was performed by flow cytometry using validated assays in CellCarta. Related analysis and related statistics

[0293] Biomarkers from the exploratory endpoint were analyzed for their associations with each other and with the efficacy endpoint. Spearman's rank correlation was used to assess the associations between analytes. Kaplan-Meir plots and Cox regression were used to assess the association between biomarkers and time to event endpoints. The Wilcoxon rank-sum test and logistic regression were used to assess the relationship between biomarkers and binary outcomes. The Kruskal-Wallis test was used to assess the association between biomarkers and categorical endpoints. For these post-hoc analyses, all p-values ​​were descriptive, and P<.05 was considered significant. No adjustments were made for multiple studies. Covariates were defined by median, quartile, or as indicated (e.g., 3721mm). 2 The data was subdivided into subgroups based on the SPD value. Plots were generated using TIBCO Spotfire, SAS, R, or GraphPad Prism. Example 2

[0294] This example demonstrates that axi-cel (autologous anti-CD19 chimeric antigen receptor T-cell therapy) as a second-line therapy significantly improved event-free survival compared to the standard care arm in patients with early relapsed or primary refractory large B-cell lymphoma (R / R LBCL) in the international Phase 3 ZUMA-7 trial.

[0295] Patients were randomized in a 1:1 ratio to either axi-cel or standard care (chemoimmunotherapy defined by a 2-3 cycle protocol, followed by high-dose chemotherapy with autologous stem cell transplantation [HDT-ASCT] in responding patients). A primary intention-to-treatment analysis of overall survival was performed for each protocol 5 years after randomization of the first patients.

[0296] A total of 359 patients were randomized to either axi-cel (n=180) or standard care (n=179). At a median follow-up of 47.2 months, axi-cel demonstrated a statistically significant improvement in overall survival compared to standard care (hazard ratio, 0.726; 95% CI: 0.540–0.977; stratified one-sided log-rank P=0.0168). The median overall survival was not achieved in the axi-cel arm and was 31.1 months in the standard care arm; estimated 48-month overall survival rates were 54.6% and 46.0%, respectively. The survival benefit in favor of axi-cel was consistent across pre-identified key subgroups, including those aged 65 years or older, with primary refractory or high-grade B-cell lymphoma. The median progression-free survival, as assessed by the principal investigator, was 14.7 months with axi-cel compared to 3.7 months with standard care, with estimated 48-month rates of 41.8% vs. 24.4%, respectively (hazard ratio, 0.506; 95% CI: 0.383~0.669). Axi-cel as a second-line treatment for R / R LBCL significantly improved overall survival compared to standard care.

[0297] For nearly 30 years, the standard care paradigm for second-line treatment of large B-cell lymphoma (LBCL) aimed at cure has been multi-step platinum-based chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation (HDT-ASCT) for patients who respond to this approach. However, it is likely that only half of all patients are eligible for this approach, and of those, approximately 20% ultimately achieve a cure. Outcomes for patients who cannot proceed to HDT-ASCT are poor, with a median overall survival (OS) of 4.4 months.

[0298] Given this unmet need and the approval of chimeric antigen receptor (CAR) T-cell therapy in third-line or later settings, the Phase 3 ZUMA-7 trial (NCT03391466) was designed to compare secondary standard care with axi-cel, an anti-CD19 autologous CAR T-cell therapy administered as a single dose, in patients with early relapsed or primary refractory (R / R) LBCL. The primary endpoint of event-free survival (EFS), as determined by a blinded central review, demonstrated that axi-cel was superior to standard care (hazard ratio [HR], 0.398; stratified log-rank P<0.0001). With a median follow-up of 24.9 months, the median EFS was 8.3 months versus 2.0 months and the EFS rates were 24 months versus 41% versus 16% in the axi-cel versus standard care arms, respectively. A blinded, centralized review showed a response rate of 83% in the axi-cel arm and 50% in the standard care arm (with complete responses of 65% and 32%, respectively). We hereby report the primary OS analysis of ZUMA-7 at a time defined by the protocol, 5 years after the initial randomization of patients. method Test design and supervision

[0299] Adult patients (18 years of age or older) had histologically confirmed LBCL that was refractory to first-line treatment or had relapsed after complete response within 12 months of first-line chemoimmunotherapy.

[0300] Patients were randomized in a 1:1 ratio to either axi-cel or standard care (HDT-ASCT in patients with complete or partial response after 2-3 cycles of chemoimmunotherapy as defined by the investigator's protocol). Randomization was stratified by response to first-line therapy and secondary age-adjusted international prognosis index (aaIPI). Crossover between treatment arms was not planned per protocol, but patients were able to receive subsequent off-treatment protocols, including cellular immunotherapy, after standard care (defined as a treatment switch). Endpoints and evaluation

[0301] The primary endpoint of ZUMA-7 was EFS (time to progression, death, or new lymphoma treatment) as determined by a blinded central review. Protocol-defined key secondary endpoints included a blinded central review and objective response rate per OS (time to death from any cause). Secondary endpoints included progression-free survival (PFS; time to disease progression or death from any cause) and EFS (reported herein according to investigator assessment, with blinded central reviews discontinued for each protocol after the primary EFS analysis). Disease assessments were performed at 50, 100, and 150 days after randomization, then every 3 months until 2 years, and every 6 months until 5 years of follow-up. statistical analysis

[0302] A pre-specified intention-to-treatment (ITT) primary OS analysis should have been performed after the first patient was randomized and triggered by the latter criterion, or within 5 years after death in 210 patients. A group sequential trial procedure for OS was performed to control for a 2.5% overall one-sided alpha. A log-rank test stratified by randomization stratification factors was used for primary comparisons of OS at the efficacy boundary with a one-sided significance level of 0.0249. In addition to the ITT analysis, two pre-specified OS sensitivity analyses were performed to adjust for confounding effects of switching treatment from the standard care arm to off-protocol cell immunotherapy.

[0303] Efficacy analyses based on the ITT principle included all randomized patients. Safety analyses included all randomized patients who received ≥1 dose of axi-cel or standard care for each protocol. All adverse events (AEs) were reported from the earlier of a hospital visit or change in lymphoma treatment, from randomization to 150 days post-randomization. Targeted serious AEs were reported from 150 days until the data cutoff date, until the first occurrence of disease progression or initiation of a new lymphoma treatment. Serious AEs assessed by the principal investigator as being related to axi-cel were reported regardless of the time of occurrence. Further methods research treatment

[0304] Patients in the axi-cel arm should receive cyclophosphamide (500 mg / m³) 5, 4, and 3 days prior to receiving a single axi-cel infusion after leukocytapheresis. 2 ( / day) and fludarabine (30 mg / m²) 2 The patient received lymphocyte depletion chemotherapy using (target dose, 2 × 10) / day. 6 Chimeric antigen receptor [CAR] T cells / kg). Glucocorticoids can be administered as optional bridging therapy. Patients in the standard care arm received two or three cycles of protocol-defined platinum-based chemoimmunotherapy. Patients who demonstrated a complete or partial response progressed to high-dose chemotherapy with autologous stem cell transplantation. Endpoints and evaluation

[0305] Event-free survival (EFS) was defined as the best response to stable disease up to that point, including the time from randomization to the earliest date of disease progression according to the Lugano classification, initiation of a new treatment for lymphoma, death from any cause, or response at the 150-day assessment after randomization. Safety outcomes included the incidence of adverse events (AEs). Guidelines for the management of CAR T-cell-related AEs followed those used in ZUMA-1. AEs, including cytokine release syndrome (CRS) and neurological events, were graded according to the National Cancer Institute Common Terminology Criteria for AEs version 4.03. The severity of CRS was graded according to the modified Lee criteria. Reporting of serious adverse events

[0306] Targeted serious adverse events were defined as neurological, hematological, infectious, autoimmune disorders, and secondary malignancies, and included those first reported for the standard care arm or the axi-cel arm, up to 5 or 15 years, or until disease progression occurred. Serious adverse events that researchers assessed as being related to axi-cel were reported regardless of the time period. Analysis of overall survival

[0307] For each group-by-group sequential trial procedure using a pre-specified rho-family consumption function to control for a total one-sided alpha of 2.5%, overall survival (OS) could be tested up to three times. According to the protocol, the first interim analysis of OS was performed at the time of the primary EFS analysis. The second interim analysis of OS was to be performed when approximately 160 deaths had been observed since the initial patient was randomized, or within four years. Approximately 160 deaths had been observed at the time of the primary EFS analysis. Therefore, the provisional OS analysis performed at the time of the primary EFS analysis was the only provisional analysis that met the criteria for both initially planned provisional OS analyses. The only subsequent planned OS analysis was the primary OS analysis reported herein, which was planned to occur when 210 death events had been observed, or within five years of the initial patient's randomization, and occurred five years after the initial patient's randomization (because fewer than 210 deaths had occurred at the time defined by that protocol). Exploratory analysis

[0308] Exploratory analyses were conducted to determine the relationship between OS and axi-cel pharmacokinetics and product characteristics. Anti-CD19 CAR T cell levels in the blood were quantified, and product T cell phenotypes and other attributes were assessed as previously described. Variables were characterized using the median (i.e., ≤median and >median) as cutoff points for comparison.

[0309] The presence of B cells in the blood was assessed for 24 months using flow cytometry in an axi-cel arm, as described above. B cell dysplasia was defined as undetectable B cell levels (i.e., below the lower limit of quantification of the assay used [less than 0.017% of leukocytes]). B cell recovery was defined as detectable B cell levels compared to the previous time point.

[0310] Long-term cytopenia was retrospectively evaluated over four time intervals and defined as present at 6, 12, 18, and 24 months after the initiation of curative treatment in the protocol (i.e., after the first dose of axi-cel infusion or high-dose therapy). Additional statistical methods

[0311] As previously reported, Kaplan-Meier estimates were provided for the time to event endpoint. Estimated hazard ratios with two-sided 95% confidence intervals were calculated from a Cox proportional hazards model stratified by a randomization stratification factor. Stratified log-rank p-values ​​(one-sided) were calculated for time to the event endpoint.

[0312] To adjust for confounding effects of switching to off-protocol cellular immunotherapy in the standard care arm, pre-identified OS sensitivity analyses were performed using two validated methods: a rank-preserved structure failure time (RPSFT) model with g-estimation and inverse probability of monitoring weight (IPCW). Patients in the standard care arm who received cellular immunotherapy were not censored at the time of cellular immunotherapy. In this model, survival / mortality times in standard care patients after cellular immunotherapy were reduced as if cellular immunotherapy had not been performed.

[0313] The association between product characteristics or levels of CAR T cells and efficacy endpoints was investigated by post-hoc univariate analysis, and descriptive p-values ​​were reported. No adjustments were made for multiple trials. Covariates were classified as continuous or using their median. Hazard ratios were calculated by a Cox regression model as the rate of increase in hazard for a one-unit increase in a continuous variable. Stratified (derived) Cox proportional hazards p-values ​​were calculated. result patient

[0314] Between January 25, 2018, and October 4, 2019, 359 patients were enrolled and randomized to either axi-cel (n=180) or standard care (n=179). Baseline patient demographics and disease characteristics were similar across treatment arms and consistent with the real-world patient population receiving CAR-T cell therapy (Table 7). Common high-risk characteristics included high-grade B-cell lymphoma (19% HGBL; including double-hit lymphoma) as assessed by the principal investigator, 45% with high second-line aaIPI scores (2 or 3), 54% with elevated lactate dehydrogenase levels, and 74% with disease resistant to first-line treatment.

[0315] [Table 7] Effectiveness

[0316] During a median follow-up period of 47.2 months (range 39.8–60.0), 82 patients in the axi-cel arm and 95 patients in the standard care arm died. A primary analysis of OS demonstrated a statistically significant improvement in OS in the axi-cel arm compared to standard care (HR for death, 0.726; 95% CI: 0.540–0.977; stratified one-sided log-rank P=0.0168). The median OS (95% CI) was not achieved in the axi-cel arm (28.6 months–not estimable [NE]) and was 31.1 months (17.1–NE) in the standard care arm (Table 8). The estimated OS rate at 48 months was 54.6% (95% CI, 47.0–61.6) in the axi-cel arm and 46.0% (38.4–53.2) in the standard care arm (Table 9). The OS benefit of axi cel over standard care was consistent across key pre-specified high-risk subgroups, including age 65 years or older, primary refractory disease, high second-line treatment aaIPI, and HGBL (including double-hit lymphoma) (Table 10).

[0317] [Table 8]

[0318] [Table 9]

[0319] [Table 10]

[0320] In the standard care arm, 102 patients (57%) received subsequent cellular immunotherapy off protocols due to progression or lack of response (Table 11). A preliminary sensitivity analysis designed to assess the confounding effects of treatment switching on OS in the standard care arm showed a greater OS benefit with axi-cel compared to standard care (Table 12).

[0321] [Table 11]

[0322] [Table 12]

[0323] PFS assessed by the principal investigator confirmed the benefit of axi-cel over standard care, with a median PFS of 14.7 months (95% CI, 5.4–43.5) for axi-cel and 3.7 months (95% CI, 2.9–5.3) for standard care (HR, 0.529 [95% CI, 0.383–0.669]; descriptive one-sided P<0.0001) (Table 13). The estimated PFS rate at 48 months was 41.8% (95% CI, 34.1–49.2) for axi-cel and 24.4% (95% CI, 17.2–32.2) for standard care. The median EFS assessed by the principal investigator differed from the primary endpoint of EFS per central review, being 10.8 months (95% CI, 5.0–25.5) for axi-cel and 2.3 months (95% CI, 1.7–3.1) for standard care, 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).

[0324] Tables 13 and 14 below show progression-free survival and event-free survival as assessed by the investigators. Table 13 shows the Kaplan-Meier estimate of progression-free survival as assessed by the investigators, defined as the time from randomization to disease progression or death from any cause. Table 14 shows the Kaplan-Meier estimate of EFS (defined as the time from randomization to the earliest date of disease progression according to the Lugano classification, initiation of a new treatment for lymphoma, or death from any cause) as assessed by the investigators. For both Tables 13 and 14, data were censored for patients who did not meet the event criteria. Stratified Cox regression models were used to provide estimated hazard ratios and two-sided 95% confidence intervals for axi-cel relative to standard care hazard ratios. The sleepers of the Cox regression models were handled using Breslow's method. One-sided p-values ​​from log-rank tests are reported. axi-cel is axicaptagensilolucel, EFS, event-free survival; HR, hazard ratio; PFS, progression-free survival.

[0325] [Table 13]

[0326] [Table 14] safety

[0327] The safety analysis set included 170 patients treated with axi-cel and 168 patients receiving standard care. All patients reported at least one therapeutic adverse event (TEAE); cumulative grade-free TEAEs, grade ≥3 TEAEs, and serious TEAEs are shown in Tables 15 and 16, respectively. In the safety analysis set, 74 patients in the axi-cel arm and 91 patients in the standard care arm died after the start of the study.

[0328] [Table 15]

[0329] [Table 16]

[0330] Disease progression was the most common cause of death in both the axi-cel (n=51) and standard care arm (n=71) (Table 17). The mortality rate associated with curative treatment (axi-cel or HDT-ASCT) was 1 / 170 (1%) in the axi-cel arm and 2 / 64 (3%) in the standard care arm (Table 17). No cumulative treatment-related serious or fatal AE changes occurred after the primary EFS analysis.

[0331] [Table 17]

[0332] New or secondary malignancies were reported in 11 patients (8 in axi-cel, all of whom were assessed by the principal investigators as being unrelated to axi-cel). Three standard care regimens were followed since the start of the study (including one patient with two new malignancies) (Table 18). No cases of replication-eligible retrovirus infection were reported.

[0333] [Table 18]

[0334] In the axi-cel arm, infections of any grade and grade 3 or higher were reported in 76 patients (45%) and 28 patients (16%), respectively, and in the standard care arm, in 53 patients (32%) and 20 patients (12%), respectively (Table 19). Of the 162 axi-cel treated patients whose B cell levels were evaluated, 47% had B cell dysplasia (undetectable B cells) up to 3 months after infusion (Table 20; Table 21).

[0335] [Table 19]

[0336] [Table 20]

[0337] [Table 21]

[0338] B-cell recovery (detectable B-cell levels compared to previous time points) was observed over time with wide inter-patient variability. The median B-cell level remained below the lower limit of quantification (0.017%) until 6 months post-infusion, and began to increase at 9 months, which coincided with the disappearance or very low levels of CAR T cells in the blood (median around 0.1 cells / μL; see Table 21, above).

[0339] Hypogammaglobulinemia was reported in 11% and 1% of patients in the axi-cel arm and the standard care arm, respectively; all cases were grade 1-2 (Table 22). Twenty-eight patients (16%) in the axi-cel arm received intravenous immunoglobulin therapy at the discretion of the researchers (Table 22).

[0340] [Table 22]

[0341] Long-term cytopenia, thrombocytopenia, and neutropenia of grade ≥3 were reported in 8 patients (5%), 0 patients (0%), and 6 patients (4%), respectively, in the axi-cel arm (N=170) 6 months after the initiation of curative treatment, and in 1 patient (2%), 1 patient (2%), and 0 patients (0%), respectively, in the standard care arm (n=62) (Table 23).

[0342] [Table 23]

[0343] Following the initial EFS analysis (Table 15, see above), no new CRS or neurological events were reported in any of the treatment arms. Exploratory translation analysis

[0344] Peak CAR T cell levels and area under the curve within the first 28 days after injection were not significantly associated with overall survival (OS) (Tables 24 and 25). In patients treated with axi-cel, the OS benefit was unrelated to the characteristics of the axi-cel product, with two notable exceptions (Table 26).

[0345] Tables 24 and 25 show the association between overall survival and CAR T cell proliferation. Kaplan-Meier estimates and estimated hazard ratios for overall survival in axi-cel patients, based on post-infusion CAR T cell peak levels (defined as the maximum number of CAR T cells in the blood after axi-cel infusion) (Table 24) and AUC0–28 (Table 25). CAR T cell proliferation was subgrouped based on median. Stratified Cox regression models were used to provide estimated hazard ratios and two-sided 95% confidence intervals for median ≤ median relative to axi-cel peak > peak. The sleepers of the Cox regression model were handled using Breslow's method. One-sided p-values ​​from log-rank tests are shown. AUC0–28 is the area under the curve from day 0 to day 28, axi-cel is axicaptagensilolucel, CAR is chimeric antigen receptor, HR is hazard ratio; OS is overall survival.

[0346] [Table 24]

[0347] [Table 25]

[0348] [Table 26]

[0349] In the analysis of OS versus product characteristics, improved OS was associated with a larger proportion of younger or stem memory T cell phenotypes (CCR7+CD45RA+ T cells) in the axi-cel product (stratified description P=0.0085) (not shown). Conversely, worse OS was associated with a larger proportion of differentiated T cells, particularly effector memory (CCR7-CD45RA-), in the axi-cel product (stratified description P=0.0091) (not shown). Table 27 below shows various product characteristics of the axi-cel product. More specifically, increased overall survival was observed in patients with an increased percentage of younger or stem memory T cell phenotypes (i.e., naive T cells) relative to the median for younger or stem memory T cells in the axi-cel product, while decreased overall survival was observed in patients with an increased percentage of differentiated T cells relative to the median for differentiated T cells in the axi-cel product.

[0350] [Table 27] Further results Pre-planned provisional OS analysis results

[0351] As previously reported, a pre-planned interim analysis of OS was performed at the time of the primary EFS analysis in ZUMA-7. In the intention-to-treatment analysis set, the median OS was not achieved in the axi-cel arm and was 35.1 months in the standard care arm (hazard ratio [HR], 0.73; 95% CI, 0.53–1.01), supporting axi-cel. In accordance with the U.S. Food and Drug Administration's request for additional survival follow-up of discontinued patients, the interim OS analysis was updated to include information from public records prior to the data cutoff date of March 18, 2021. In that analysis, the median OS was not achieved in the axi-cel arm and was 25.7 months in the standard care arm (HR, 0.71; 95% CI: 0.52–0.97). Pre-specified sensitivity analysis of OS

[0352] In a pre-specified sensitivity analysis designed to evaluate the confounding effect of treatment switching on OS in the standard care arm, axi-cel showed significantly longer median OS than standard care using a rank-preserved structure-deficient time model (not reached [95% CI, 28.6 months–not estimable] vs. 15.5 months [95% CI, 9.7–not estimable]; HR, 0.608 [95% CI, 0.449–0.824]; stratified log-rank one-sided description P=0.0006) (Table 12). Similar results were obtained using a weight-limited inverse stochastic method (HR, 0.633; 95% CI: 0.438–1.118). Additional response outcomes

[0353] In this analysis, the objective response rate per investigator was 83% (complete response rate 61%) in the axi-cel arm and 45% (complete response rate 34%) in the standard care arm. 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 group and the standard care arm, respectively. At the data cutoff, 71 / 180 (39%) and 29 / 179 (16%) patients, respectively, showed an ongoing response. Additional safety results

[0354] Two patients had ongoing neurological events at the time of the previous analysis: one patient randomized to the axi-cel arm had grade 2 paresthesia and grade 1 memory impairment, both symptoms persisting until the patient's death; and one patient randomized to the standard care arm had grade 1 paresthesia, which remained ongoing during long-term follow-up. Consideration

[0355] In this trial comparing two second-line treatment strategies for R / R LBCL patients, the risk of death with axi-cel was significantly lower by 27.4% compared to standard care, and the 4-year survival rate was absolutely improved by 8.6%.

[0356] Prior to the advent of CAR-T cell therapy, second-line LBCL patients who could not progress to curative treatment with HDT-ASCT had poor outcomes, with a median OS of 4.4 months. Previous attempts to improve patient survival in the second-line curative situation were unsuccessful, suggesting that the maximum benefit of a chemotherapy-based approach had been achieved. The most recent trial to demonstrate improved survival was the 1995 Parma trial, conducted before the approval of rituximab. Therefore, a new non-chemotherapy-based second-line approach was needed in high-risk patients with early R / R LBCL, which prompted the design of the ZUMA 7 study.

[0357] Axi-cel was the first CAR-T cell therapy to receive regulatory approval for second-line therapy of LBCL in the United States, the European Union, and many other countries, based on superior EFS and response with axi-cel compared to chemotherapy / HDT-ASCT in ZUMA-7. The results of the ZUMA-7 primary OS analysis reported herein clearly demonstrate that patients treated with axi-cel have a clear survival advantage over patients treated with second-line platinum-based chemotherapy and, if responsive, with HDT-ASCT. Importantly, the stability of the OS and PFS Kaplan-Meier survival curves up to 4 years suggests the curative potential of second-line axi-cel for a significant proportion of patients.

[0358] The OS benefit of axi-cel over chemotherapy / HDT-ASCT was consistent across key pre-specified patient subgroups known to have poor prognoses, including patients aged 65 years and older, patients with disease resistant to first-line therapy, and patients with HGBL. The benefit of Axi-cel in elderly patients is particularly interesting because patients considered ineligible for curative treatment with HDT-ASCT due to age may still be eligible for CAR T-cell therapy. Since the improvements in OS and EFS reported with axi-cel in elderly patients were at least similar to those in the overall population, axi-cel may expand the patient population that can benefit from treatment intent.

[0359] Furthermore, axi-cel was associated with a significant OS benefit in more than half of the patients in the standard-care arm, despite subsequent off-protocol cell immunotherapy due to lack of response or disease progression. Notably, this proportion was similar to other modern randomized anti-CD19 CAR T-cell therapy trials, including protocol-specific cross-designs. Historically, standard-care patients with early relapse or prior rituximab treatment showed estimated 3-year OS rates of 39% and 40%, respectively, while patients with both early relapse and prior rituximab, representing the ZUMA 7 patient population, had a 3-year survival rate of less than 40%. In contrast, standard-care patients in ZUMA-7 showed an estimated 3-year OS rate of 48%. Given the increased survival rates in the standard-care arm compared to previous studies, due to the availability of third-line CAR T-cell therapy during ZUMA-7, the true survival benefit of second-line axi-cel over standard care may be even greater, as supported by our treatment switching analysis. Based on the clearly improved survival rates compared to platinum-based chemotherapy / HDT-ASCT, axi-cel should be the standard second-line treatment, as opposed to attempting second-line chemotherapy and only administering cellular immunotherapy when it proves to be inadequate.

[0360] The long-term safety profile of axi-cel was consistent with previous studies. Notably, according to the protocol, the AE reporting period ended with disease progression or initiation of new lymphoma treatment, both of which were disproportionately high in the standard care arm. Long-term cytopenia and immunodeficiency, including B-cell dysplasia and induction of infection, are expected with CD19-targeted CAR T-cell therapy, representing on-target / off-tumor class effects. In particular, the incidence of prolonged grade 3 or higher cytopenia began at 6 months post-axi-cel infusion and decreased over time. Given the occurrence of grade 3 or higher infections, hypogammaglobulinemia, and B-cell dysplasia with axi-cel, clinical monitoring of patients treated with CAR-T-cell therapy is important to mitigate the risk of long-term infection. B-cell recovery was observed over time in the majority of patients treated with axi-cel, suggesting that the lasting clinical benefit does not depend on the long-term persistence of functional CAR T cells, as previously described for axi-cel in third-line or later settings.

[0361] In addition to improved survival rates, axi-cel was associated with improved quality of life (QoL). Patients treated with Axi-cel had significantly longer periods of symptom- or toxicity-free life, clinically significant increases in quality-adjusted survival rates, clinically meaningful improvements in QoL, and faster recovery to baseline compared to standard care.

[0362] Importantly, OS is an objective endpoint unaffected by observer bias, and the ZUMA-7 primary OS analysis clearly establishes that the second-line axi-cel treatment strategy for early R / R LBCL is superior to chemotherapy followed by HDT-ASCT in responding patients. With platinum chemotherapy as the first-line second-line treatment, only a small number of patients receive curative treatment with HDT-ASCT, mainly due to a lack of chemotherapeutic agent sensitivity (unknown before treatment initiation). The improvement in OS with axi-cel highlights the importance of early consultation with axi-cel before initiating second-line chemotherapy.

[0363] ZUMA-7 demonstrated a significant improvement in survival with axi cel compared to second-line chemotherapy / HDT-ASCT in patients with R / R LBCL, and long-term outcomes were consistent with curative therapy. Example 3

[0364] This example discloses a classification model for identifying patients at risk of neurotoxicity and CRS after axicaptagen silolucel (axi-cel) treatment for second-line R / R LBCL.

[0365] Axi-cel is an autologous anti-CD19 CAR T-cell therapy approved for relapsed / refractory (R / R) large B-cell lymphoma (LBCL) after one or more lines of systemic therapy. Its superiority over standard care in second-line treatment (refractory or early relapse) was demonstrated in the randomized phase 3 Zuma-7 trial. Post-axi-cel cytokine release syndrome (CRS) and neurological events (NE) were reported in 92% and 61% of patients, respectively; high-grade (grade 3+) events were reported in 6% and 21%, respectively. While the pathophysiology of CRS is well-established, the underlying mechanisms of NE remain unclear. We are interested in prospectively identifying patients at low risk of toxicity who can be treated in an outpatient setting.

[0366] The objective here was to construct a data-driven multivariate classification model in secondary R / R LBCL using pretreatment biomarkers to identify patients with the following toxicity-related outcomes: Grade 3+ (G3+) NE at any point after axi-cel, and low grade or no toxicity (maximum Grade 1 CRS and no NE within 7 days after axi-cel). The inventors also sought to validate superior models in multiple R / R LBCL populations, as identified in Table 28 below.

[0367] The inventors incorporated approximately 450 biomarker + time point combinations. Candidate biomarkers included demographics (approximately 5), clinical and disease characteristics (approximately 30), routine serological chemistry and hematology (approximately 30), cytokines and other pharmacodynamic markers (approximately 25), and product attributes (approximately 30). Time points for measuring these biomarkers were included: baseline (i.e., before lymphocyte depletion), day 0 (before Axi-Cel treatment); and the change multiplier from baseline to day 0, calculated as (day 0) / (baseline). Machine learning methods (e.g., conditional random forest, XGBoost) were used to select features. A logistic classifier was trained using the selected features, and its performance was evaluated on a holdout test set (70:30% split). Variability was estimated using bootstrap resampling. A data-driven approach was implemented (i.e., the estimated set of clinically relevant biomarkers was not pre-selected). A clinician-curated model was selected from top data-driven models.

[0368] [Table 28]

[0369] Table 29 shows the results of top models for predicting grade 3+ neurotoxicity identified using a data-driven approach. Selection was based on high area under receiver operating characteristics (AUROC), high negative predictive value (NPV), and consistent covariate orientation across training and test sets. In Table 29, higher levels of biomarkers are associated with a higher likelihood of grade 3+ neurotoxicity development.

[0370] [Table 29]

[0371] Table 30 shows the results for the top data-driven models with low grade or no toxicity. Selection was based on high AUROC, high PPV, and consistent covariate orientation for the training and test sets. In Table 30, higher levels of biomarkers are associated with a higher likelihood of low or no toxicity.

[0372] [Table 30]

[0373] While predicting which patients will experience high-grade NE toxicity remains a challenge, these results demonstrate good performance (i.e., high NPV) in predictively identifying a subset of patients less likely to experience high-grade NE toxicity. The disclosed results include a best-performing classifier for low-grade or no toxicity (defined by grade 1 or less CRS and no NE), achieving a 70% PPV (7 out of 10 correctly classified) in training and test sets, suggesting a possible means of identifying patients for outpatient administration. This modeling work is useful to assist clinicians in prospectively managing patients at risk of toxicity, as well as those for whom outpatient monitoring may be desirable. Example 4

[0374] Axi-cel is an autologous anti-CD19 CAR T-cell therapy approved for relapsed / refractory (R / R) follicular lymphoma (FL). Approval was supported by the Phase 2, multicenter, single-arm ZUMA-5 trial of xi-cel in patients with R / R low-grade non-Hodgkin lymphoma (iNHL; N=104) (including FL and marginal zone lymphoma [MZL]). In the primary analysis (median follow-up period of 17.5 months), the overall response rate (ORR) was 92% (complete response rate 74%). Here, we report the long-term outcomes from ZUMA-5. Eligible patients with R / R iNHL after two or more treatments received leukocytapheresis followed by lymphocyte depletion chemotherapy and axi-cel infusion (2 × 10⁶). 6Patients received CAR T cells ( / kg). The primary endpoint was ORR, which was assessed by the investigators in all enrolled patients (intention to treat) in this analysis. After a median follow-up of 41.7 months (n=127) in FL and 31.8 months (n=31) in MZL, ORR was comparable to the primary analysis (94% in FL; 77% in MZL). The median progression-free survival was 40.2 months in FL and had not yet been reached in MZL. Median overall survival was not reached in either disease subtype. Grade 3 or greater adverse events of interest occurred because previous analyses were primarily in recently treated patients. Clinical and pharmacokinetic outcomes were negatively correlated with recent exposure to bendamustine and high metabolic tumor volume. After 3 years of follow-up of ZUMA-5, axi-cel demonstrated a sustained response, with few relapses beyond 2 years, and showed manageable safety in R / R iNHL patients. Introduction

[0375] Relapsed or refractory (R / R) low-grade non-Hodgkin lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL), is largely considered incurable, with most patients eventually experiencing further disease relapses. While subsequent treatment outcomes are heterogeneous among FL patients, a common thread is progressively shorter remissions and decreased survival rates after second-line and subsequent therapies. Furthermore, FL patients who progress within 24 months of initiating initial anti-CD20-containing chemoimmunotherapy (POD24) have an unfavorable prognosis and shortened survival time, despite the availability of R / R treatment options.

[0376] Recent advances in treatment options for iNHL, including chimeric antigen receptor (CAR) T-cell therapy, are improving outcomes for patients with R / R disease. Axi-cel is an autologous anti-CD19 CAR T-cell therapy containing a CD28 costimulatory domain that delivers target-specific cytotoxicity and induces rapid and robust proliferation that helps overcome immune system limitations. Axi-cel is approved for the treatment of adults with R / R FL. Approval was supported by a primary analysis of the ZUMA-5 trial, a single-arm international phase 2 trial in iNHL patients (N=104), which showed an overall response rate (ORR) of 92% (74% complete response [CR] rate) after a median follow-up of 17.5 months.

[0377] Long-term follow-up analyses are particularly important in R / R low-grade lymphoma due to the heterogeneity of pre-treatment tumor characteristics and their long clinical course. Here, we report the efficacy, safety, and biomarker evaluations of ZUMA-5 after 3 years of follow-up, which represents the longest follow-up analysis of CAR T-cell therapy in iNHL to date. This analysis includes an exploratory assessment of the association between clinical outcomes and baseline variables (including baseline systemic tumor tissue volume assessed by prior bendamustine exposure and metabolic systemic tumor tissue volume (MTV)). method Patient and research design

[0378] ZUMA-5 is a multicenter, single-arm, enrollment, phase 2 trial conducted at 17 medical centers in the United States and France, and is registered at Clinicaltrials.gov / NCT03105336. A complete list of sites has been previously reported. Participating patients provided written informed consent to participate, and the trial protocol was approved by the institutional review board at each site.

[0379] The complete patient eligibility criteria have been previously reported. Briefly, patients aged 18 years or older with R / R iNHL, including FL (grades 1-3a) and MZL (nodular or extranodal); both according to WHO 2016 criteria, had undergone at least two prior systemic therapies, which had to include anti-CD20 monoclonal antibodies in combination with alkylating agents. Patients who had undergone autologous stem cell transplantation (SCT), any allogeneic SCT, CD19 targeted therapy, or CAR T-cell therapy within 6 weeks of axi-cel were excluded. Disease progression less than 6 months after completion of the most recent prior therapy was considered refractory. Procedures and endpoints

[0380] Registered patients received fludarabine (30 mg / m²) 5 to 3 days prior to the infusion. 2 ( / day) and cyclophosphamide (500 mg / m²) 2 / day) and axi-cel(2×10 6 I underwent lymphocyte depletion chemotherapy using CAR T cells ( / kg). 9 Bridge therapy before lymphocyte depletion was at the discretion of the principal investigator. Disease response assessments were performed by the researchers as previously detailed, and reviewed by an independent radiological review committee according to the Lugano classification (previously detailed) at specified points up to the 24-month follow-up analysis, after which assessments were performed solely by the researchers. All adverse events (AEs) were monitored up to 3 months post-infusion, and then only AEs of particular interest (neurological, hematological, infectious, and autoimmune) were monitored up to 24 months. New and secondary malignancies were monitored up to 15 years.

[0381] The primary endpoint of ZUMA-5 was ORR. Secondary endpoints included CAR T-cell complete response (CR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), time to next treatment (TTNT), safety, and blood levels. Exploratory endpoints included in this analysis were lymphoma-specific PFS and survival, with progression events or deaths associated with lymphoma, axi-cel, or lymphocyte depletion chemotherapy, as assessed by the researchers, considered events of interest. Clinical and pharmacokinetic outcomes were also evaluated by key patient and clinical subgroups, including prior bendamustine use before leukocytosis and baseline MTV.

[0382] statistical analysis The 3-year analysis of ZUMA-5 was performed when enrolled FL patients had a median follow-up of 36 months or more. Efficacy outcomes were evaluated in all enrolled patients with iNHL (intent to treat). Safety and translational evaluations were performed in patients treated with iNHL (laboratory and biomarker evaluations are described previously). FL patients with three or more lines of treatment were evaluated in separate analyses, except for patients with alternating histology at baseline median assessment. Patients retreated with axi-cel were also evaluated separately (retreatment criteria are reported previously).

[0383] Descriptive statistics were used to summarize baseline characteristics, responses, and incidence of adverse events (AEs). Two-sided 95% confidence intervals (CIs) for objective response rates were assessed using the Clopper-Pearson method. Secondary endpoints, including time to event outcomes, were assessed using the Kaplan-Meier method. Lymphoma-specific PFS and survival were assessed using a competing risk approach, where the event of interest was considered the primary event and death not attributable to lymphoma, axi-cel, or lymphocyte depletion chemotherapy was considered a competing risk. Event rates over time were calculated for both the primary event and competing risks via a cumulative incidence function.

[0384] Propensity score matching (PSM) was performed to assess outcomes in FL patients with prior bendamustine use, explaining baseline MTV, ECOG score, FLIPI score, number of previous chemotherapy regimens, age, distribution of dual refractory status, and whether the last systemic therapy was leukocytapheresis for <12 months. The Wilcoxon rank-sum test was used to assess the association between CAR T cell levels and clinical outcomes. result patient

[0385] A total of 159 patients were enrolled (127 with FL, 31 with MZL, and 1 with DLBCL), including 6 additional MZL patients who received leukocytapheresis and were enrolled after the data cutoff date for 18-month analysis. Axi-cel was successfully manufactured for all enrolled patients. In addition to the previously described untreated patients, one patient had disease transformation and one patient did not have a measurable disease. Patients determined to have DLBCL did not receive axi-cel and were discontinued from the study. A total of 152 patients had received prior chemotherapy and axi-cel as of the data cutoff date of March 31, 2022 (124 with FL and 28 with MZL).

[0386] Baseline characteristics for all 159 registered patients are not shown. The median age was 60 years (range, 34-79) for FL patients and 64 years (range 43-77) for MZL patients. Among FL patients, 56% had POD24 and 69% had prior bendamustine use. Table 31 shows the baseline characteristics of FL patients based on prior bendamustine exposure before and after PSM.

[0387] [Table 31]

[0388] *Propensity score matching was performed using accurate descriptions of PTR12MFL after 1:1 matching with log2 MTV caliper=1 and age caliper=1.5. †One observation regarding the number of previous treatment lines was missing before and after matching in patients exposed to bendamustine for 12 months prior to leukocytapheresis or less. Eight observations for POD24 were missing before and after matching in patients without prior bendamustine exposure. Two observations for POD24 were missing before and after matching in patients exposed to bendamustine for 12 months prior to leukocytapheresis or less. ECOG PS is the performance status of the US East Coast Cancer Clinical Trials Group; FLIPI is the International Prognostic Index for Follicular Lymphoma, IQR, interquartile range; mAb is monoclonal antibody, MTV, metabolic tumor volume; POD24 is disease progression less than 24 months after initiation of initial anti-CD20 formulation.

[0389] Baseline MTV in FL patients positively correlated with total product diameter (SPD; not shown), FLIPI score, and systemic tumor tissue volume as measured by GELF criteria, but did not correlate with baseline lactate dehydrogenase levels. Efficacy in patients with follicular lymphoma

[0390] The median follow-up period for leukocytapheresis in enrolled FL patients was 41.7 months (range, 32.7–57.4). Detector-assessed responses among enrolled FL patients were consistent with previous analyses (ORR, 94% [95% CI, 88–97]; CR rate, 79%). The median duration of response (DOR) in patients with FL was 38.6 months. The median DOR was not achieved in CR patients and was 4.9 months in patients with partial response. At the data cutoff, 53% (67 / 127) of enrolled FL patients had an ongoing response. Of those who achieved CR (n=100), 65% had an ongoing response at the data cutoff. Consistent with previous reports, all 13 patients with FL responded to retreatment with axi-cel (69% CR; 31% PR). At a median of 23 months after retreatment, the median duration of response (DOR) after retreatment was 5.0 months, and 46% of patients showed an ongoing response at the data cutoff.

[0391] The median PFS for enrolled FL patients was 40.2 months, and the estimated 36-month PFS rate was 54%. A total of 2 progression events and 10 deaths occurred beyond 24 months after leukocytapheresis. In the competing risk analysis for lymphoma-specific PFS in FL patients, a total of 40 progression or death events occurred (31%) due to lymphoma, lymphocyte depletion chemotherapy, or axi-cel, of which 38 were progression events. The 36-month cumulative incidence of lymphoma-specific progression or death was 34%. Competing risk (death excluding progression or investigational treatment) occurred in a total of 13 patients (10%), most occurring after 24 months. The cumulative incidence of competing risk at 36 months was 12%. The median PFS for patients with POD24 (n=70) or those without POD24 (n=41) was 40.2 months, respectively, and was not reached. The estimated 36-month PFS rate was nearly consistent in FL patients, regardless of other high-risk baseline characteristics.

[0392] FL patients who had previously received bendamustine had a numerically lower 36-month PFS rate compared to patients who had not received bendamustine. Patients who received bendamustine ≤12 months of leukocytapheresis had a numerically significantly shorter PFS, although the small number of patients in this group may limit the comparison (Table 32; Table 33). Further examination of prior bendamustine use after PSM showed numerically higher CR and ongoing response at 36 months in patients who had not been previously exposed to bendamustine compared to patients who had been exposed to ≤12 months of leukocytapheresis (Table 34). Notably, these findings were not statistically significant, and these analyses generate hypotheses, which were likely powerless.

[0393] Table 32 shows progression-free survival (PFS) for follicular lymphoma patients based on the timing of bendamustine use prior to Axi-Cel infusion. Kaplan-Meier estimates of progression-free survival for enrolled FL patients, as assessed by researchers, are categorized as: no prior bendamustine exposure ("none"), bendamustine administered within 6 months of leukapheresis ("<6 months"), bendamustine administered for 6 to 12 months ("6 to 12 months"), and bendamustine administered more than 12 months before leukapheresis (">12 months"). axi-cel represents axicaptagensilolucel, FL represents follicular lymphoma, NE represents not estimable, PFS, and progression-free survival.

[0394] [Table 32]

[0395] [Table 33]

[0396] CR: Complete response; DOR: Duration of response; NE: Not estimable; NR: Not reached; ORR: Overall response rate; PFS: Progression-free survival; OS: Overall survival.

[0397] [Table 34]

[0398] Propensity score matching was performed using accurate descriptions of PTR12MFL after 1:1 matching with log2 MTV caliper=1 and age caliper=1.5. AUC is area under the curve, CAR is chimeric antigen receptor, CR is complete response; CRS is cytokine release syndrome; IFN is interferon, IQR is interquartile range; MTV is metabolic tumor volume; ORR is overall response rate.

[0399] The median OS among enrolled FL patients was not reached, and the estimated OS at 36 months was 76%. The median TTNT was also not reached, with an estimated TTNT of 60% at 36 months. Competitive risk assessment for lymphoma-specific OS showed 15 deaths (12%) due to lymphoma, lymphocyte depletion chemotherapy, or axi-cel. Competitive risk (death from other causes) occurred in 17 patients (13%). The 36-month cumulative incidence of lymphoma-specific death was 12% (the cumulative incidence of competing risk at 36 months was 12%).

[0400] In evaluating efficacy outcomes by baseline MTV in evaluable patients with FL (n=125), DOR and PFS were longer in patients with relatively low baseline MTV, as observed below the historical threshold of 510 mL, and below the study median and interquartile (Tables 35, 36, and 37). In particular, the estimated PFS at 36 months was 71.2% in patients with baseline MTV below the study median, compared to 37.3% in patients with baseline MTV above the median. No correlation was observed between median baseline MTV and ORR or CR, which is likely due to the small number of non-responders in this study. Patients with baseline MTV below the study median were also more likely to be in an ongoing response at the data cutoff than patients with baseline MTV above the median. The association between baseline SPD and efficacy outcomes showed a similar trend to MTV, but did not reach statistical significance.

[0401] Table 35 shows progression-free survival in FL patients by quartile of baseline metabolic tumor volume. Kaplan-Meier plot of progression-free survival per investigator-assessed by quartile of baseline metabolic tumor volume in evaluable enrolled FL patients. FL stands for follicular lymphoma, NE stands for not estimable, PFS, and progression-free survival.

[0402] [Table 35]

[0403] [Table 36]

[0404] CR: Complete response; DOR: Duration of response; NE: Not estimable; NR: Not reached; ORR: Overall response rate; PFS: Progression-free survival.

[0405] [Table 37]

[0406] CR: Complete response; DOR: Duration of response; NE: Not estimable; NR: Not reached; ORR: Overall response rate; PFS: Progression-free survival.

[0407] Efficacy results are reported separately in a subset of FL patients with three or more prior treatment lines, after excluding patients whose central pathological assessment suggested an alternative diagnosis other than FL. The results were in close agreement with the overall cohort. Effectiveness in patients with marginal zone lymphoma

[0408] The median follow-up period for enrolled patients with MZL following leukocytapheresis was 31.8 months (range 8.3–52.3). The investigator-assessed ORR in enrolled patients with MZL was 77% (95% CI, 59–90), and the CR rate was 65%. Responses between MZL subtypes (nodular and extranodal) were not shown. The median DOR for all MZL patients has not yet been reached, and 52% (16 / 31) of patients had an ongoing response at the data cutoff.

[0409] The median PFS, OS, and TTNT have not yet been reached among MZL patients, with 24-month estimates of 56%, 74%, and 53%, respectively. 24-month PFS estimates were fairly consistent among the high-risk subgroups. No correlation was observed between baseline MTV and efficacy outcomes among MZL patients, likely due to the small number of patients with this disease subtype in the study (Table 36 above). safety

[0410] No new safety signals were observed among patients treated for iNHL after the 18-month analysis. AEs occurring after the 18-month analysis (data cutoff date, September 14, 2020) were mostly in recently enrolled MZL patients and included one grade 3 neurological event, two grade 3–4 infections, and five grade 3–4 cytopenia. Serious AEs occurred in 15 patients (10%; 11 in FL and 4 in MZL from the 18-month analysis); events in 6 of these patients were considered axi-cel related (3 in FL and 3 in MZL). No new cases of grade ≥3 hypogammaglobulinemia occurred after the primary analysis data cutoff date (March 12, 2020). Furthermore, there were no new secondary malignancies since the 18-month analysis. During the course of the study, a total of 50 iNHL patients (33%) received immunoglobulin therapy. No cases of axi-cel-related secondary malignancies, oncolytic syndromes, or retroviruses with replication ability occurred at any point in the study.

[0411] When evaluating all events in the iNHL trial, Grade 3 or higher cytokine release syndrome cases appeared to occur more frequently in patients aged 65 and older (12%) than in patients under 65 (4%). Similarly, Grade 3 or higher neurological events occurred numerically more frequently in patients aged 65 and older (27%) than in patients under 65 (14%). Of the iNHL patients (n=51) who had any grade ≥3 cytopenia 30 days or later post-infusion, 5 had cytopenia present at 12 months post-infusion and 4 had cytopenia at 24 m...

Claims

1. A method for predicting the likelihood of a response to a cell therapy product in a patient who needs it, 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 is quantified. To determine, at least partially, the likelihood of the patient responding to the cell therapy product based on the gene expression levels, Includes, An increase in the gene expression level of at least one gene compared to a control value indicates the possibility of an increased or decreased response, compared to a predetermined probability of the response rate. The gene expression level is quantified from the patient sample, and the patient sample is collected from the patient before treatment with the cell therapy product. A method for predicting the likelihood of response to cell therapy products in patients who require them.

2. The method according to 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 an increase in the gene expression level of the at least one gene compared to a control value indicates an increase in the probability of response compared to a predetermined probability of response rate.

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

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

5. The method according to 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 an increase in the gene expression level of the at least one gene compared to a control value indicates a decrease in the likelihood of response compared to a predetermined likelihood of response rate.

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

7. The method according to 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 according to claim 7, wherein the cell therapy product is autologous or allogeneic.

9. The target antigen is preferably a tumor-associated surface antigen, such as 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, β-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, C D30, CD33, CD34, CD4, CD40, CD44, CD56, CD79a, CD79b, CD123, FLT3, BCMA, SLAMF7, CD8, CLL-1, c- Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III ( EGFRvIII), ELF2M, Endoglin, Ephrin B2, Epidermal Growth Factor Receptor (EGFR), Epithelial Cell Adhesion Molecules (EpCAM), Epithelial Tumor Antigen, ErbB2 (HER2 / neu), Fibroblast-Associated Protein (fap), FLT3, Folate-Binding Protein, GD2, GD3, Glioma-Associated Antigen, Sphingoglycolipid, gp36, HBV-Specific Antigen, HCV-Specific Antigen, HER1-HER2, HER2-HER3 Combinations, HERV-K, High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), HIV-1 Envelope Glycoprotein gp41, HPV-Specific Antigen, Human Telomerase Reverse Transcriptase, IGF-II Receptor, IGF-II, IL-11R-alpha, IL-13R-a2, Influenza Virus-Specific Antigen;CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, κ chain, LAGA-1a, λ chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific antigen or tissue-specific antigen, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecule, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, variant hsp70-2, variant p53, variant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 The method according to claim 7, wherein the tumor antigen is selected from antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivorin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigen, e.g., HIV-specific antigen (e.g., HIV gp120), GPC3 (glypican 3), and any derivative or variant of these antigens.

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

11. The aforementioned patients have solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (non-specific 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 myeloid leukemia, acute leukemia One or more of the following: 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"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, pilocytic cell leukemia Diseases, small cell or large cell follicular lymphoma, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal dysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance (MGUS), plasmacytoplasmic lymphoma The method according to any one of claims 1 to 10, wherein the patient is diagnosed with a cancer / tumor selected from the group consisting of ma cell tumors (e.g., plasma cell proliferation disorder, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndromes (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 carcinoma, prostate cancer, breast cancer, or a combination thereof.

12. The method according to claim 11, wherein the cancer is (recurrent or refractory) diffuse large B-cell lymphoma (DLBCL) nonspecific type, mediastinal large B-cell lymphoma, high-grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

13. The method according to any one of claims 1 to 12, wherein the cell therapy product is selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagenlecleucel, lysocabbutagen maralucel, and bb2121.

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

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

16. A method for treating malignant tumors in patients, 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 is quantified. At least partially, by quantifying the gene expression level of at least one gene, it is possible to determine whether an effective dose of cell therapy product should be administered to a patient as a second-line therapy or as a third-line therapy, Based on the above determination, the effective dose of the cell therapy product is administered as a second-line therapy or a third-line therapy. Includes, The gene expression level is quantified from the patient sample, and the patient sample is collected from the patient before treatment with the cell therapy product. If the expression level of at least one of the genes CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is equal to or greater than the control value for at least one of the genes, the patient is administered the effective dose of the cell therapy product as a second-line therapy, or If the expression level of at least one of the genes CD19, MS4A1, TNFRSF17, BLK, FCRL2, FAM30A, PNOC, SPIB, and TCL1A is below the 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, or If the expression level of at least one of the following genes is below the control value for at least one of the following genes, the patient is administered the effective dose of the cell therapy product as a second-line therapy, or If the expression level of at least one of the following genes is greater than or equal to the control value for at least one gene, the patient is administered the effective dose of the cell therapy product as a third-line therapy. Methods for treating malignant tumors in patients.

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

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

20. The method according to 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 according to claim 20, wherein the cell therapy product is autologous or allogeneic.

22. The target antigen is preferably a tumor-associated surface antigen, such as 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, β-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 antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant II I (EGFRvIII), ELF2M, Endoglin, Ephrin B2, Epidermal Growth Factor Receptor (EGFR), Epithelial Cell Adhesion Molecules (EpCAM), Epithelial Tumor Antigen, ErbB2 (HER2 / neu), Fibroblast-Associated Protein (fap), FLT3, Folate-Binding Protein, GD2, GD3, Glioma-Associated Antigen, Sphingoglycolipid, gp36, HBV-Specific Antigen, HCV-Specific Antigen, HER1-HER2, HER2-HER3 Combinations, HERV-K, High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), HIV-1 Envelope Glycoprotein gp41, HPV-Specific Antigen, Human Telomerase Reverse Transcriptase, IGF-II Receptor, IGF-II, IL-11Rα, IL-13R-α2, Influenza Virus-Specific Antigen;CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, κ chain, LAGA-1a, λ chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific antigen or tissue-specific antigen, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecule, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, variant hsp70-2, variant p53, variant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate-carcinoma tumor antigen-1 The method according to claim 20, wherein the tumor antigen is selected from antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecule, survivorin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigen, e.g., HIV-specific antigen (e.g., HIV gp120), GPC3 (glypican 3), and any derivative or variant of these antigens.

23. The method according to claim 20, wherein the cell therapy product expresses a chimeric antigen receptor containing a CD28 costimulatory domain.

24. The aforementioned patients have solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), mediastinal large B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) (non-specific 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 myeloid leukemia, acute leukemia One or more of the following: 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"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, pilocytic cell leukemia Lymphoma, small cell or large cell follicular lymphoma, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal dysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance (MGUS), plasma The method according to any one of claims 16 to 23, wherein the patient is diagnosed with a cancer / tumor selected from the group consisting of cell tumors (e.g., plasma cell proliferation disorder, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), 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 carcinoma, prostate cancer, breast cancer, or a combination thereof.

25. The method according to claim 24, wherein the cancer is (recurrent or refractory) diffuse large B-cell lymphoma (DLBCL) nonspecific type, mediastinal large B-cell lymphoma, high-grade B-cell lymphoma (HGBL), DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

26. The method according to any one of claims 16 to 25, wherein the cell therapy product is selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagenlecleucel, lysocabbutagen maralucel, and bb2121.

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

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