Prediction of adverse events originating from immunotherapy

Abstract

The present invention relates to diagnostic methods and prognosis prediction methods, compositions for immunotherapy, methods for improving said compositions, and immunotherapies using said compositions (e.g., T cell, non-T cell, TCR-based therapies, CAR-based therapies, bispecific T cell engagers (BiTEs), and / or immune checkpoint blockers). [Solution] A method for determining the risk of adverse events during immunotherapy administration to cancer patients requiring immunotherapy, comprising: quantifying the baseline serum level of one or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 310,438, filed on 15 February 2022, the entire contents of which are incorporated herein by reference.

[0002] (Field of Invention) This disclosure relates to diagnostic methods and prognosis prediction methods, compositions for immunotherapy, and immunotherapy using the same. [Background technology]

[0003] Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformations, resulting in abnormal cancer cells. This causes cancer cells to begin expressing proteins (including, but not limited to, antigens) that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T lymphocytes and B lymphocytes, from normally targeting them.

[0004] Human T cell therapy is based on human T cells that have been enriched or modified to target and kill a patient's cancer cells. Methods have been developed to genetically engineer T cells to express constructs that direct T cells toward specific target cancer cells, thereby increasing the ability of T cells to target and kill specific cancer cells. For example, chimeric antigen receptors (CAs) containing binding domains that can interact with specific tumor antigens. R) and T cell receptors (TCRs) allow T cells to detect the specific tumor antigen. This allows for the targeting and elimination of expressing cancer cells. It is necessary to understand how pre-treatment patient attributes (e.g., serum proteomics profile, systemic tumor tissue volume, tumor genetic profile, and proteomics profile) influence treatment outcomes, including the safety outcomes of immunotherapy. [Overview of the Initiative]

[0005] It should be understood that the uses of this disclosure are not limited to the embodiments, claims, description, and drawings described below. Other embodiments of this disclosure are possible, and it can be implemented or performed in numerous other ways.

[0006] In this specification, specific parameters after administration of immunotherapy (e.g., cell therapy), such as complete response (CR) or partial response (PR), are used. Methods are provided for evaluating the expression of specific biomarkers or analytes that may correlate with outcomes such as therapeutic outcomes, which include responses, or safety outcomes such as toxicity, for example, neurotoxicity or the expression of CRS. Methods are also provided for evaluating the likelihood of response and / or the likelihood of toxicity risk based on parameters, for example, 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 immunochecks) 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, for example, leukemia or lymphoma. A chokepoint blocker is provided. In some embodiments, the method and use improve the response and / or the efficacy of a particular alternative method compared to a subject treated by several methods. To provide or achieve a more sustained response or efficacy and / or a reduced risk of toxicity or other adverse effects. In some embodiments, the method includes administering a specific number or relative number of manipulated cells, administering a predetermined ratio of a specific type of cell, treating a specific patient population such as a patient population with a specific risk profile, stage classification, and / or prior treatment history, administering additional therapeutic agents, and / or combinations thereof.

[0007] In one embodiment, the disclosure provides that baseline (preconditioning) serum levels of specific 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, metabolic process biomarkers are PAG1, SOD2, and KYNU. In one embodiment, leukocyte activation biomarkers are AREG, NUB1, HLA-DRA, LY9, SERNPINB9, OSMR, IL1A, REG3A, and REG1B. In one embodiment, biomarker levels are analyzed by an Olink panel. 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 tumor-infiltrating lymphocyte (TIL) immunotherapy The treatment is selected from autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. In a particular embodiment, eACT is engineered antigen-specific chimeric antigen receptor (CAR)-positive (+) T cells. This includes the administration of cells. In another embodiment, eACT includes the administration of engineered antigen-specific T cell receptor (TCR)-positive (+) T cells, in one embodiment, immunotherapy. This is CAR T cell therapy or TCR T cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T cell therapy.

[0008] In one embodiment, the present disclosure shows that baseline (preconditioning) serum levels of specific proteins associated with the IL1 / IL6 pathway are positively correlated with Grade 3+ cytokine release syndrome (CRS) after immunotherapy, and that their biomarkers —Provides that it may be possible. In one embodiment, the biomarkers are IL1A and OSMR. In one embodiment, the disclosure provides that baseline (preconditioning) serum levels of certain proteins associated with inflammatory endothelial markers may be positively correlated with both Grade 3+ CRS and Grade 3+ neurological events (NEs) after immunotherapy and may be their biomarkers. In one embodiment, the inflammatory endothelial markers are ACE2, CEACAM1, and ICAM 2 , and ADAM15. 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 therapy or TCR T-cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T-cell therapy.

[0009] In one embodiment, the present disclosure provides that the baseline serum levels of MET, OSMR, ACE2, ACY1, and BILDIR are positively correlated with grade 3+ CRS and NE after immunotherapy and may be their biomarkers. In one embodiment, the present disclosure provides that the baseline serum levels of CCL16, CELA3A, MEGF9, MFAP3, REG1B, AREG, BSG CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM2, LY9, REG3A, SERPINB9, SOD2, ADA2, ITM 2

[0010]

[0011] In one embodiment, the present disclosure provides that the baseline serum levels of GPA33, EPCAM, and CRNN are positively correlated with grade 3+ CRS and NE after immunotherapy and may be their biomarkers. In one embodiment, the present disclosure provides that the fold change values between the day 0 serum levels and the baseline serum levels of GPA33, EPCAM, and CRNN are positively correlated with grade 3+ CRS and NE after immunotherapy and may be their biomarkers. In one embodiment, the serum levels of one or more of these proteins are positively correlated with preconditioning and / or immune activation-induced stress after preconditioning regimens that are exacerbated by immunotherapy and may be their biomarkers. In one embodiment, the preconditioning regimen includes cyclophosphamide and fludarabine. The following are non-limiting embodiments of the present disclosure.Embodiments of this disclosure are methods for determining the risk of adverse events during immunotherapy administration to cancer patients requiring immunotherapy, the methods comprising quantifying baseline serum levels from cancer patients of one or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET, or SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRR The present invention relates to a method comprising: quantifying the day-0 serum level of one or more biomarkers selected from EL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5 from a cancer subject; and determining the risk of adverse events in a cancer subject based on the steps of quantifying the baseline serum level of one or more biomarkers or quantifying the day-0 serum level of one or more biomarkers from a cancer subject. In such embodiments, at least one of the following is correlated with an increased risk of adverse events in a cancer subject: an increase in the baseline serum level of one or more biomarkers relative to the control baseline serum level of one or more biomarkers and / or an increase in the day-0 serum level of one or more biomarkers relative to the control day-0 serum level of one or more biomarkers. In such embodiments, the adverse events are one or more of the following: immune activation-induced stress, grade 3+ CRS, and grade 3+ NE. In such embodiments, baseline serum levels refer to serum levels of one or more indicated biomarkers, and serum samples were collected before administration of a preconditioning regimen to the cancer subject. In such embodiments, day 0 serum levels refer to serum levels of one or more indicated biomarkers, and serum samples were collected after administration of conditioning therapy to the cancer subject, but before administration of immunotherapy to the cancer subject.

[0012] One embodiment of the present disclosure is that the method quantifies the baseline serum levels of one or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET from a cancer subject, and SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5, quantifies the serum levels on day 0 from a cancer subject, and determines the risk of adverse events in a cancer subject based on the step of quantifying the baseline serum levels of one or more biomarkers and the step of quantifying the serum levels on day 0 from a cancer subject of one or more biomarkers.

[0013] One embodiment of the present disclosure relates to any of the above methods, wherein the method includes quantifying the baseline serum levels of two or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET from a cancer subject.

[0014] An embodiment of the present disclosure relates to any of the above methods, wherein the method includes quantifying the serum levels on day 0 from a cancer subject of two or more biomarkers selected from SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5.

[0015] Embodiments of the present disclosure relate to any of the above methods, wherein the control baseline serum level of one or more biomarkers is a baseline serum level observed as a history of one or more biomarkers that has not been previously observed to be associated with the development of an adverse event, and the control day 0 serum level of one or more biomarkers is a day 0 serum level observed as a history of one or more biomarkers that has not been previously observed to be associated with the development of an adverse event.

[0016] Embodiments of the present disclosure relate to any of the above methods, wherein KYNU quantifies baseline (preconditioning) serum levels of one or more biomarkers from cancer subjects, and KYNU quantifies day 0 serum levels of one or more biomarkers from cancer subjects, selected from ACE2, ADAM15, BSG, CEACAM1, HLA-DRA, ICAM2, LY9, LY96, OSMR, REG3A, SERPINB9, SOD2, and VAMP5.

[0017] Embodiments of this disclosure include baseline serum levels of one or more biomarkers selected from MET, OSMR, NELL2, REG3A, KYNU, and AREG, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCHD17, STK11, TGFB1, ACE2, ADAM15, BSG, CEACAM1, HLA-DRA, ICAM2, LY9, LY96, OSMR, REG3A, SERPINB9, SOD2, VAMP5, CCL16, CELA3A, MEGF9, MF If the day 0 serum level of one or more biomarkers selected from AP3 and REG1B exceeds the specified normalized protein expression range for one or more biomarkers, cancer patients are determined to have an increased risk of adverse events. The specified normalized protein expression ranges for one or more biomarkers are 0.56-0.75 for MET, 0.62-0.77 for OSMR (baseline), 0.14-0.50 for NELL2, 1.80-2.19 for REG3A (baseline), and 1.60- for KYNU. 1.98, 0.84~1.25 for CCL16, 1.37~2.01 for CELA3A, 0.26~0.48 for MEGF9, 0.33~0.49 for MFAP3, 1.49~1.99 for REG1B, 0.88~1.22 for AREG, 0.73~0.96 for BSG, 1.83~2.31 for CKAP4, 3.48~3.91 for CXCL1, 0.09~0.23 for EIF5A, 0.46~0.81 for IL1A, 1.36~1.66 for KIFBP, and for KIRREL2... For 1.62~2.13, for NUB1 1.39~1.77, for OSMR (day 0) 0.65~0.79, for PAG1 2.16~2.74, for PCDH17 1.03~1.32, for STK11 1.09~1.35, for ACE2 1.20~1.65, for ADAM15 1.09~1.31, for CEACAM1 0.68~0.90, for HLA-DRA 1.13~1.41, for ICAM2 1.21~1.43, for LY9 0.11~0.49, for REG3A (day 0) 2 The above methods relate to one of the following ranges: 0.34~2.88 for SERPINB9, 2.12~2.30 for SOD2, 1.37~1.86 for LY96, 0.54~0.70 for TGFB1, 1.51~1.67 for TGFB1, and 1.30~1.73 for VAMP5. In such embodiments, the normalized protein expression range is calculated, at least in part, based on the mean, standard deviation (SD), and standard error (SE) of the expression data for each respective biomarker. In such analysis, "0" refers to the group with no toxicity, and "1" refers to the group with toxicity. SE is calculated as SD / sqrt(n), where "n" represents the sample size in each group. The lower and upper limits are set as Mean.NPX+ / -1 * Calculations are performed as SE (for the toxic group). Each of the calculated ranges shown above for each of the biomarkers mentioned represents a reasonable range for possible cutoff points for each biomarker, and observed values ​​exceeding the cutoff point indicate that cancer subjects may experience Grade 3+ cytokine release syndrome and / or Grade 3+ neurological events within 5 days after immunotherapy treatment.

[0018] Embodiments of the present disclosure relate to any of the above methods, further comprising, when a cancer subject is determined to be at increased risk of adverse events, one or more of the following: treating the cancer subject with a preconditioning regimen modified from a standard preconditioning regimen; not administering immunotherapy to the cancer subject; administering immunotherapy to the cancer subject at a dose modified from a standard dose; or administering immunotherapy to the cancer subject in combination with an agent for mediating immune activation-inducing stress, grade 3+ CRS, and / or grade 3+ NE.

[0019] Embodiments of the present disclosure relate to any of the above methods, wherein the modified preconditioning regimen includes administering a modified dose of at least one of cyclophosphamide and fludarabine to a cancer subject instead of a predetermined dose of cyclophosphamide or fludarabine administered to a control subject.

[0020] Embodiments of the present disclosure relate to any of the above methods, further comprising administering immunotherapy in combination with a combination therapy including a drug that reduces cytokine induction and / or endothelial cell destruction when a cancer patient is determined to be at high risk of adverse events.

[0021] Embodiments of this disclosure relate to any of the above methods for which a drug reduces cytokine induction.

[0022] Embodiments of this disclosure relate to any of the above methods, wherein the drug is administered to a cancer target before administration of immunotherapy, before the peak expansion of immunotherapy, or during the peak expansion of immunotherapy.

[0023] Embodiments of the present disclosure relate to any of the above methods, wherein the agent is selected from anti-IL-1 molecules, T cell activation inhibitors, JAK inhibitors, anti-GM-CSF molecules, anti-TNF molecules, Ang2 inhibitors, anti-angiogenic therapies, and anti-IFNg molecules.

[0024] Embodiments of this disclosure relate to any of the above methods, wherein the immunotherapy is CAR T cell therapy, TCR T cell therapy, tumor-infiltrating lymphocyte (TIL) cell therapy, or bispecific T cell engager (BiTE) therapy.

[0025] Embodiments of this disclosure relate to any of the above methods, wherein the immunotherapy is autologous or allogeneic.

[0026] Embodiments of this disclosure describe immunotherapy in which the immunotherapy is CAR T cell therapy or TCR therapy that recognizes a target antigen. This relates to one of the above methods of T-cell therapy.

[0027] Embodiments of this disclosure preferably use tumor-associated surface antigens, 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, and CD3 4, 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, tubular 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-associated protein (fap), FLT3, leaf Acid-binding proteins, GD2, GD3, glioma-associated antigens, sphingoglycolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight-melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific 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 carboxylester -ase, 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) 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 (PCT) A-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 A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, blood Vascular endothelial growth factor receptor-2 (VEG) The present invention relates to any of the above methods, wherein the tumor antigen is selected from FR2), a virus-specific surface antigen, such as an HIV-specific antigen (e.g., HIV gp120), GPC3 (glypican 3), and any derivative or variant of these antigens.

[0028] Embodiments of this disclosure include cancers such as 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), DLBC arising from FL L, transformed follicular lymphoma, high-grade B-cell lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoid leukemia ("BALL"), one or more of the above, T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (AL L), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia 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 gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., plasma cell dyscrasia, 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 cancer, prostate cancer, breast cancer, or a combination thereof, related to any of the above methods.

[0029] Embodiments of the present disclosure relate to any of the above methods where the cancer is (recurrent or treatment-resistant) diffuse large B-cell lymphoma (DLBCL) - unspecified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

[0030] Embodiments of the present disclosure relate to any of the above methods where the immunotherapy is selected from axicabtagene ciloleucel, brexucabtagene autoleucel, tisagenlecleucel, lisocabtagene maraleucel, and bb2121.

[0031] Embodiments of the present disclosure relate to a method for determining the risk of adverse events during immunotherapy administration to a cancer subject requiring immunotherapy, the method comprising: determining baseline serum levels and / or day 0 serum levels of a plurality of biomarkers from the cancer subject; determining weights for each of the plurality of biomarkers, at least partially, by inputting the baseline serum levels of the plurality of biomarkers into an algorithm; and determining the probability of a risk value for an adverse event, at least partially based on the weights for each of the plurality of biomarkers. In such embodiments, if the probability of the risk value is at least as large as a predetermined cutoff value, the subject has an increased risk of adverse events compared to a control group, and the plurality of biomarkers include ACE2, IL1A, SERPINB9, and LY96. In such embodiments, baseline serum levels refer to serum levels of one or more indicated biomarkers, and serum samples are collected before administration of a preconditioning regimen to the cancer subject. In such embodiments, day 0 serum levels refer to serum levels of one or more indicated biomarkers, and serum samples are collected after administration of conditioning therapy to the cancer subject but before administration of immunotherapy to the cancer subject.

[0032] Embodiments of this disclosure relate to the above method, wherein the predetermined cutoff is a Youden cutoff value of 0.10 to 0.35.

[0033] Embodiments of this disclosure relate to any of the above methods, wherein the algorithm includes a logical regression model, a random forest algorithm, or a regularized gradient boosting framework algorithm.

[0034] Further embodiments of this disclosure are as follows:

[0035] When administering immunotherapy to cancer patients requiring immunotherapy, inflammation-induced stress A method for predicting grade 3+ CRS and / or grade 3+ NE, wherein the method comprises pretreatment, (i) Baseline (preconditioning) serum levels of one or more biomarkers selected from ACE2, ACY1, OSMR, BILDIR, and MET. (ii) Serum levels of one or more biomarkers selected from REG1B, MEGF9, CCL16, CELA3A, and MFAP3 on day 0 (after conditioning and before immunotherapy), (iii) Serum levels on day 0 (after conditioning and before immunotherapy) of one or more IL1A, REG3A, ICOSLG, ITM2A, CGA, VAMP5, REG1B, LY9, HLA-DRA, LY96, CEACAM1, SOD2, ADA2, BSG, EIF5A, MEGF9, OSMR, PCDH17, ADAM15, ICAM2, SERPINB9, CCL16, KIRREL2, ACE2, AST, CELA3A, MFAP3, followed by the remaining AREG, CKAP4, CXCL1, KIFBP, NUB1, PAG1, and STK11. (iv) Baseline (preconditioning) serum levels of one or more biomarkers related to the IL1 / IL6 pathway (e.g., IL1A and OSMR), and (v) Baseline (preconditioning) serum levels of specific proteins associated with inflammatory endothelial markers (e.g., CEACAM1, ACE2, ICAM2, and ADAM15), (vi) Quantifying the value of the fold change between the day 0 serum level and the baseline serum level of CRNN, EPCAM, and GPA33, The higher the serum levels of these protein biomarkers, or the greater the fold change between baseline and day 0 serum levels, the higher the immune activation-inducing stress and / or the higher the likelihood that the subject will develop grade 3+ CRS and / or grade 3+ NE after immunotherapy. (vii) If a patient has lower baseline serum levels of FGF21 or one or more of CGA, ICOSLG, or ITM2A on day 0, the patient is more likely to develop grade 3+ CRS and / or grade 3+ NE after immunotherapy.

[0036] MET, OSMR, ACE2, ACY1, FGF21, BILDIR; Day 0: CCL16, CELA3A, MEGF9, MFAP3, REG1B, AREG, BSG Baseline serum levels of one or more biomarkers selected from CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM2, LY9, REG3A, SERPINB9, SOD2, ADA2, ICOSLG, ITM2A, CGA, CRNN, LY96, AST, and VAMP5, and the fold change between day 0 and baseline for GPA33, EPCAM, and CRNN are as clearly indicated, with the following values ​​for each of the proteins: baseline 0.56-0.75 (MET, 0.62-0.77 (OSMR, 0.65-0.98 (ACE2, 0.66-1.10 (ACY1, 0.66-1.10 (ACY1, 0.66-2.25 ( FGF21, lower), 4.12~5.50 (BILDIR, upper), day 0 0.84~1.25 (CCL16, upper), 1.37~2.01 (CELA3A, upper), 0.26~0.48 (MEGF9, upper), 0.33~0.49 (MFAP3, upper), 1.49~1.99 (REG1B, upper), 0.88~1.22 (AREG, upper), 0.73~0.96 (BSG, upper), 1.83~2.31 (CKAP4, upper) , 3.48~3.91 (CXCL1, upper), 0.09~0.23 (EIF5A, upper), 0.46~0.81 (IL1A, upper), 1.36~1.66 (KIFBP, upper), 1.62~2.13 (KIRR EL2, top), 1.39~1.77(NUB1, top), 0.65~0.79(OSMR, top), 2.16~2.74(PAG1, top), 1.03~1.32(PCDH17, top), 1.09~1.35( STK11, upper), 1.20~1.65 (ACE2, upper), 1.09~1.31 (ADAM15, upper), 0.69~0.90 (CEACAM1, upper), 1.13~1.41 (HLA-DRA, upper), 1.21~1.43 (ICAM2, upper), 0.11~0.49 (LY9, upper), 2.34~2 .88 (REG3A, top), 2.12~2.30 (SERPINB9, top), 1.37~1.86 (SOD2, top), 0.70~0.98 (AD A2, upper), 0.74~1.01 (ITM2A, lower), 0.45~0.56 (ICOSLG, lower), 1.12~1.50 (CGA, upper), -0.4 The above method is used to determine whether a subject has a higher or lower baseline serum level than a subject with baseline serum levels below or above the listed values, such as 8-0.08 (CRNN, upper), 0.54-0.70 (LY96, upper), 42.12-65.80 (AST, upper), and 1.30-1.73 (VAMP5, upper), as well as a divergence change between day 0 and baseline of -1.36-(-0.60) (GPA33, lower), -0.82-(-0.39) (EPCAM, lower), and -0.27-0.16 (CRNN, upper), and the subject has a higher likelihood of developing grade 3+ CRS and / or grade 3+ NE after immunotherapy than a subject with baseline serum levels below or above the listed values, respectively. In some such embodiments, the listed baseline serum levels are measured by Olink. In some such embodiments, subjects are more likely to develop grade 3+ CRS and / or grade 3+ NE after immunotherapy than subjects with baseline serum levels below or above the median baseline serum level, respectively. In such embodiments, changes between baseline and day 0 biomarker levels may indicate systemic dysregulation in the subject as a result of any conditioning chemotherapy regimen, which may then make the subject more susceptible to toxicity after administration of immunotherapy (e.g., CAR T-cell therapy).

[0037] The above method, using measured serum levels of biomarkers, to guide subsequent decisions related to immunotherapy administered to a subject, including, but not limited to, whether, what type of immunotherapy, what immunotherapy dosage / dosing regimen, what conditioning protocol, and / or which drugs(s) should be administered to the patient before, after, and / or during immunotherapy (e.g., CAR T cells), in order to improve management in the subject and / or reduce grade 3+ CRS and / or grade 3+ NE.

[0038] The above method, wherein the greater the change in one or more levels or magnification of biomarkers, the more conservative the conditioning regimen and immunotherapy, etc.

[0039] A method for stratifying patients who are more likely to develop high-grade adverse events, the method comprising administering immunotherapy in combination with an agent(s) that reduces immunoactivation and / or endothelial cell destruction, wherein the combination therapy reduces cytokine induction and / or endothelial cell destruction, and the patient has baseline MET, OSMR, ACE2, ACY1, BILDIR, day 0 CCL16, CELA3A, MEGF9, MFAP3, REG1B, AREG, BSG If a patient has high levels of serum markers, estimated by measuring the levels of one or more of the following proteins: CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM2, LY9, REG3A, SERPINB9, SOD2, ADA2, CRNN, LY96, AST, and VAMP5, and the fold change in CRNN in the blood between day 0 and baseline; or if a patient has low levels of serum markers, estimated by measuring the levels of one or more of the following proteins: baseline FGF21, day 0 CGA, ICOSLG, and ITM2A, and the fold change in GPA33 and EPCAM between day 0 and baseline, the patient is selected for combination therapy.

[0040] MET, OSMR, ACE2, ACY1, FGF21, BILDIR; Day 0 CCL1 6. Baseline serum levels of one or more biomarkers selected from CELA3A, MEGF9, MFAP3, REG1B, AREG, BSG, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM2, LY9, REG3A, SERPINB9, SOD2, ADA2, ICOSLG, ITM2A, CGA, CRNN, LY96, AST, and VAMP5, and the cyclic change between day 0 and baseline of GPA33, EPCAM, and CRNN. As explicitly stated, the following values ​​were obtained for each of the proteins: Baseline 0.56~0.75 (MET, top), 0.62~0.77 (OSMR, top), 0.65~0.98 (ACE2, top), 0.66~1.10 (ACY1, top), 1.58~2.25 (FGF21, bottom), 4.12~5.50 (BILDIR, top), Day 0 0.84~1.25 (CCL16, top), 1.37~2.01 (CELA3A, top), 0.26~0.48 (MEGF9, top), 0.33~0.49 (MFAP3, top), 1.49 ~1.99 (REG1B, upper), 0.88~1.22 (AREG, upper), 0.73~0.96 (BSG, upper) 1.83~2.31 (CKAP4, upper), 3.48~3.91 (CXCL1, upper), 0.09~0.23 (EIF5A, upper), 0.46~0.81 (IL1 A, upper), 1.36~1.66 (KIFBP, upper), 1.62~2.13 (KIRREL2, upper), 1.39~1.77 (NUB1, upper), 0.65~0.79 (OSMR, upper), 2.16~2.74 (PAG1, upper), 1.03~1.32 (PCDH17, upper), 1.09~1.35 (STK11, upper), 1.20~1.65 (ACE2, upper), 1.09~1.31 (ADAM15, upper), 0.69~0.90 (CEACAM1, upper), 1.13~1.41 (HLA-DRA, upper), 1.21~1.43 (ICAM2, upper), 0 .11~0.49 (LY9, upper), 2.34~2.88 (REG3A, upper), 2.12~2.30 (SERPINB9, upper), 1.37~1.86 (SOD2, upper), 0.70~0.98 (ADA2, upper), 0.74~1.01 (ITM2A, lower), 0.45~0.The above methods are used for baseline serum levels above or below the median baseline serum levels, respectively: 56 (ICOSLG, lower), 1.12-1.50 (CGA, upper), -0.48-0.08 (CRNN, upper), 0.54-0.70 (LY96, upper), 42.12-65.80 (AST, upper), and 1.30-1.73 (VAMP5, upper), as well as the fold change between day 0 and baseline: -1.36-(-0.60) (GPA33, lower), -0.82-(-0.39) (EPCAM, lower), and -0.27-0.16 (CRNN, upper). In some such embodiments, the listed baseline serum levels are measured by Olink. In some such embodiments, subjects are more likely to develop grade 3+ CRS and / or grade 3+ NE after immunotherapy than subjects with baseline serum levels below or above the median baseline serum level, respectively. In such embodiments, changes between baseline and day 0 biomarker levels may indicate systemic dysregulation in the subject as a result of any conditioning chemotherapy regimen, which may then make the subject more susceptible to toxicity after administration of immunotherapy (e.g., CAR T-cell therapy).

[0041] The above method of combination therapy reduces cytokine induction after immunotherapy.

[0042] The above method, wherein the drug(s) are administered to the patient before CAR-T infusion, before the peak of CAR-T proliferation (e.g., 0-6 days after infusion), and / or at the peak of CAR-T proliferation (e.g., 7-14 days).

[0043] The conditioning regimen / lymphocyte apheresis includes cyclophosphamide / fludarabine, bendamustine, whole-body irradiation, anti-CD45 (apamistamab), other chemotherapeutic agents (e.g., AVM0703, busulfan, thiotepa / etoposide, pe) at different doses than those of subjects with the same or lower levels of the serum biomarkers listed in claim 6 (higher levels for baseline FGF21 and day 0 CGA, ICOSLG, ITM2A), One of the above methods, selected from (e.g., antstatins).

[0044] The drug(s)

[0045] A method of treating cancer subjects with low eosinophil and / or monocyte counts on day 0 of immunotherapy (pre-immunotherapy) by administering one or more drugs or treatments that result in a reduction of inflammation. Non-limiting examples of such drugs include corticosteroids, anti-GMCSF, anti-IFNg, and anti-IFN-a (cifalimumab) and anti-IFN-b (avonex).

[0046] The method described above, wherein the subject has a low eosinophil and / or monocyte count, if the eosinophil count on day 0 is lower than (0.040, 0.065) and / or the monocyte count on day 0 is lower than (0.025, 0.055).

[0047] A method for treating a cancer subject with a high systemic tumor tissue volume, 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) before, during, and / or after immunotherapy, and / or by using an alternative lymphocyte depletion regimen (compared to that of a subject with less than a high systemic tumor tissue volume).

[0048] The reduction of inflammation is advantageous in relation to the above methods, which are beneficial for cell therapy.

[0049] Baseline whole-body tumor tissue volume (SPD) of 2500, 3000, 3500, or 4000 mm³ 2 More preferably, 3000mm 2The above method, wherein the subject has a high systemic tumor tissue volume (as evaluated 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).

[0050] A method for treating cancer subjects with a high international prognostic index (IPI), wherein immune activation-mediated stress in the subjects is reduced by administering one or more agents or treatments that result in reduced inflammation (e.g., reduced induction of cytokines in the blood) before, during, and / or after immunotherapy, and / or by using an alternative lymphocyte depletion regimen (compared to that of subjects with a high IPI or less).

[0051] The reduction of inflammation is advantageous in relation to the above methods, which are beneficial for cell therapy.

[0052] The method described above, wherein the subject has a high International Prognostic Index (IPI) when the IPI is greater than 1, preferably greater than 2, or more preferably greater than 3.

[0053] Immunotherapy is any of the above methods, including CAR T-cell therapy, TCR T-cell therapy, tumor-infiltrating lymphocyte (TIL) cell therapy, bispecific T-cell engagers (BiTEs), and / or the administration of immune checkpoint inhibitors.

[0054] Immune checkpoint inhibitors target immune checkpoint receptors on the surface of T cells, such as cytotoxic T lymphocyte antigen 4 (CTLA-4). Lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin mucin domain 3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), T cell immunoglobulin, T cell immunoreceptor suppressive tyrosine motif (ITIM) domain, and programmed cell death 1 (PD-1 / PDL-1) The above method involves selecting from drugs that block the signal.

[0055] The above-mentioned method of immunotherapy, which is either autologous or allogeneic.

[0056] The above method involves immunotherapy using CAR T or TCR T cell therapy that recognizes a target antigen.

[0057] 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 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 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 antigen, e.g., HIV-specific antigen (e.g., HIV The above method, wherein the tumor antigen is selected from gp120, GPC3 (glypican 3), and any derivative or variant of these antigens.

[0058] Cancer / tumors include solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin 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 myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), and chronic lymphocytic leukemia. One or more of the following: 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, Walde The above method, selected from Ström's macroglobulinemia, plasmacytoproliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance (MGUS), plasmacytoma (e.g., plasmacytoplasia, 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.

[0059] The cancer is (recurrent or refractory) diffuse large B-cell lymphoma (DLBCL) nonspecific type, mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, DLBCL arising from follicular lymphoma, or mantle cell lymphoma, as described above.

[0060] Immunotherapy is performed in any of the above ways, selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagen lecleucel, lysocabbutagen maralucel, and bb2121. [Brief explanation of the drawing]

[0061] [Figure 1] This is a schematic diagram showing the differences in adverse event management among ZUMA-1 cohorts.

[0062] [Figure 2] This is a series of graphs showing the correlation between Olink NPX expression and internal MSD data.

[0063] [Figure 3] This chart shows the WGCNA analysis of baseline O-Link analytes.

[0064] [Figure 4] This is a plot of baseline protein analyses associated with Grade 3+ cytokine release syndrome (CRS) (left) or Grade 3+ neurological event (NE) (right).

[0065] [Figure 5] This graph shows the baseline differentially expressed proteins in the yellow module related to responses to oxidative stress and metabolic processes.

[0066] [Figure 6] This graph shows the baseline differentially expressed proteins in the turquoise module, which is positively associated with leukocyte activation and adhesion.

[0067] [Figure 7] This chart shows the O-Link WGCNA analysis for day 0.

[0068] [Figure 8] This is a plot of day 0 protein analytes associated with Grade 3+ cytokine release syndrome (CRS) (left) or Grade 3+ neurological event (NE) (right).

[0069] [Figure 9] This graph shows the differentially expressed proteins on day 0 in the turquoise module, which is positively associated with adhesion.

[0070] [Figure 10]This graph shows the differentially expressed proteins on day 0 in the blue module related to metabolic processes.

[0071] [Figure 11] This graph shows the results of a predictive model for Grade 3+ CRS and / or NE within 5 days of CAR T injection. [Modes for carrying out the invention]

[0072] This disclosure is partly based on the finding that the proteomic properties of patients' serum before and after conditioning may be related to clinical efficacy and toxicity, including sustained responses, cytokine release syndrome of grade ≥ 3, and neurological events of grade ≥ 3. definition

[0073] 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.

[0074] 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.

[0075] As used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.”

[0076] 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).

[0077] 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.

[0078] Terms like "greater than or equal to," "at least," and "greater than," for example, "at least one," are not limiting, but rather at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 10 6, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 It is understood to include 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.

[0079] 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.

[0080] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limiting, but include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 ,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,10 5, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 It is understood to include 38, 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.

[0081] Throughout this specification, the word "comprising" or "comprises" Alternatively, variations such as “comprising” are understood to mean including the described element, integer, or process, or group of elements, integers, or processes, but not to mean excluding any other element, integer, or process, or group 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 element, process, or component 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 claims to the inclusion of materials other than those described, excluding impurities that are usually associated with them”). The term “essentially derived from” limits the scope of the claim to the materials or processes specified and “that do not substantially affect the essential and novel features (plural) of the claimed disclosure.”

[0082] As used herein, unless otherwise specified or evident from the context, the term “about” means a value or composition that lies within the acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this depends to some extent on how such value or composition is measured or determined, i.e., on the limitations 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. "Approximately" or "about" can mean a range of up to 10% (i.e., ±10%). Therefore, "approximately" can be understood as being 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, approximately 5 mg could encompass any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, this term can mean a value off by an order of magnitude or up to five times the value. Where a specific value or composition is 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 that particular value or composition.

[0083] 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.

[0084] 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.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally 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 Academic Press; "The Dictionary of Cell & Molecular Biology," 5th ed., (2013); 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 in this disclosure.

[0086] "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. Additional definitions for reference and comparison

[0087] The term "reference" describes the standard or control on which the comparison is performed. For example, in some embodiments, the protein level or gene expression level of interest is compared to a reference or control, which is a protein level or gene expression level. In certain embodiments, the reference value is from the same individual at an earlier point in time. In some embodiments, the reference or control The reference is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of the subject. In some embodiments, the reference or control is a reference or control from the past, optionally embodied in a tangible medium. In certain embodiments, the reference or control is determined or characterized within a patient population having equivalent conditions, environment, and / or characteristics of a common disease as those under evaluation. In certain further embodiments, there is sufficient similarity to justify dependence on and / or comparison with the selected reference or control.

[0088] When used throughout, the terms “baseline serum level” and “baseline (preconditioning) serum level” are used interchangeably and refer to serum levels of one or more indicated biomarkers, with serum samples collected prior to administration of the preconditioning regimen to cancer subjects.

[0089] When used throughout, the terms “control baseline serum level” and “control baseline (preconditioning) serum level” are used interchangeably and refer to baseline serum levels observed as a history of one or more analytes or biomarkers disclosed, where the baseline serum level observed as a history has not been previously observed to be associated with the development of one or more adverse events, or is associated with a reduced risk of developing one or more adverse events. In some embodiments, deviations from the baseline serum level observed as a history correlate with an increased rate of adverse events as a result of immunotherapy treatment.

[0090] When used throughout, the terms "Day 0 serum levels" and "Day 0 (post-conditioning and pre-immunotherapy)" are used interchangeably and refer to serum levels of one or more indicated analytes or biomarkers, where serum samples were collected after the administration of conditioning therapy to the cancer subject but before the administration of immunotherapy to the cancer subject.

[0091] When used throughout, the terms “control day 0 serum level” and “control day 0 (post-conditioning and pre-immunotherapy) serum level” are used interchangeably and refer to the day 0 serum level observed as a history of one or more analytes or biomarkers disclosed, where the day 0 serum level observed as a history has not been previously observed to be associated with the development of one or more adverse events, or is associated with a reduced risk of developing one or more adverse events. In some embodiments, deviations from the day 0 serum level observed as a history correlate with an increased rate of adverse events as a result of immunotherapy.

[0092] When used throughout, “control level” or “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 adverse effects to the cell therapy product. More specifically, in some embodiments, an increase in the level of an analyte in a patient-derived test sample relative to the control level for its corresponding analyte is associated with an increased likelihood of observing adverse effects in the patient as a result of administration of the cell therapy product. In such embodiments, the increased likelihood of adverse effects is measured with respect to the known historical mean likelihood of adverse effects to the cell therapy product in the population.

[0093] 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 referred to as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). A more conservative region is placed 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).

[0094] 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 Fv, scFv), camelized antibodies, affibodies, Fab Examples include 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 antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies.

[0095] 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.

[0096] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may involve either antibody production or activation of specific immune cells, or both. Those skilled in the art will readily understand that virtually all proteins or peptides, and any macromolecule, can function as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or by recombination. Antigens may be specific to certain tissues, such as cancer cells, or they may be expressed broadly. Furthermore, larger molecular fragments may act as antigens. In some embodiments, the antigen is a tumor antigen.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In one embodiment, CAR T cell therapy includes "axicabutagensilol-ucell therapy." "Axicapbutagensilol-ucell therapy" contains 2 × 10⁶ anti-CD19 CAR T cells. The treatment consists of a single intravenous infusion of autologous T cells transduced with anti-CD19 CAR at a targeted dose of T cells / kg. For subjects weighing over 100 kg, a maximum fixed dose of 2 × 10⁸ 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, exhibiting 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) undergo leukocyte apheresis and phycotherapy. Peripheral blood mononuclear cells are obtained by isolation. These cells are activated by culturing them with an anti-CD3 antibody in the presence of recombinant interleukin 2 (IL-2). The stimulated cells are transduced with a retroviral vector containing the anti-CD19 CAR gene and grown in culture to produce T cells sufficiently engineered for administration. Axicapbutagensilolucel is a target-specific product.

[0101] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into cells via a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, 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.

[0102] "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 that invade adjacent tissues and may metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer tissue" may include tumors. In this application, the term cancer is synonymous with malignant tumor. Examples of cancers that can be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein include, for example, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin 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, [add other solid tumors], multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), 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, para It may be used to reduce the tumor size of tumors resulting from combinations of the above cancers, including renal cancer, soft tissue sarcoma, urethral cancer, 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 ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and tumors resulting from 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 they may be treatment-resistant. Treatment-resistant cancers are those that are not suitable for surgical intervention, and are either unresponsive to chemotherapy or radiotherapy from the outset, or become unresponsive over time.

[0103] 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.

[0104] 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. Examples of cytokines include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, and TNF- Beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1, s ICAM-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).

[0105] Chemokines are a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include 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), and gamma-inducing chemokines. Promoting protein 10 (IP-10), as well as thymus and activation regulatory chemokines (thymus and Examples include activation-regulated chemokines (TARC or CCL17), but these Not limited to this.

[0106] 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.

[0107] 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.

[0108] 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, also called a “therapeutic agent” 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 include treatment plans in which the drugs are not necessarily administered via the same route of administration or simultaneously.

[0109] The terms “product” or “injectable product” are used interchangeably herein and do not require administration. This refers to a T-cell composition administered to a target. A typical example is CAR T-cell therapy, where the T-cell composition is administered as an infusion product.

[0110] 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 exhibit numerous functional characteristics specific to each cell type; (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 exist. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0111] In the context of this disclosure, the terms “TN,” “T naive-like,” and “CCR7+CD45RA+” actually refer to cells that are closer to stem cell-like memory cells than to typical naive T cells. Therefore, all T cells in the examples and claims are not actually T cells. N References to these cells refer to and should be interpreted as cells experimentally selected solely by their characterization as CCR7+CD45RA+ cells. A more preferred name for them in the context of this disclosure is stem cell-like memory cells, although they are referred to as CCR7+CD45RA+ cells. Further characterization of stem cell-like memory cells can be found, for example, in Arihara Y, Jacobsen CA, Armand P, et al. Journal for This can be performed using the method described in ImmunoTherapy of Cancer. 2019;7(1):P210.

[0112] 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). Sex constructs are integrated into the cell's genome.

[0113] "Immune response" is 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 these cells or the liver, against invading pathogens and cells infected with pathogens. This refers to the action of selectively targeting, binding to, damaging, destroying, and / or eliminating from the body of a vertebrate cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0114] The term "immunotherapy" refers to the treatment of individuals who have a disease or are at risk of developing or relapsing 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, and modified autologous cell therapy. Examples include 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 Applications Publications 2014 / 0154228 and 2002 / 0006409, U.S. Patents 7,741,465, 6,319,494, 5,728,388, and International Publication 2008 / 081035. In some embodiments, immunotherapy includes CAR T cell therapy. In some embodiments, the CAR T cell therapeutic product is administered via infusion.

[0115] 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 derived from, for example, peripheral blood mononuclear cells. T cells can be obtained from 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. Additional 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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 be, but is not limited to, a co-stimulatory molecule present on T cells, such as, but is not limited to, ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), NATURAL Examples include antibodies that specifically bind to tumor killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0123] "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, CD276 (B7-H3), and CD2 8, CD29, CD3(α; beta, delta, epsilon, gamma, ζ), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDDS, 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), signal transduction lymphocyte activating molecules, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleaved forms, or combinations thereof.

[0124] 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.

[0125] "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, mitigating, 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, the 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, 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). This may also include delaying the onset of one or more symptoms of a disease or condition compared to the onset of symptoms in similar untreated subjects. 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 include a judgment of not showing the applicable 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.

[0126] As used herein, “myeloid cells” refers to a subgroup of leukocytes, including granulocytes, monocytes, macrophages, and dendritic cells.

[0127] 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 common-sense methods.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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).

[0133] 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).

[0134] 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.

[0135] As used herein, the term “partial response” refers to a reduction of more than 30% of the tumor without complete recovery.

[0136] 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).

[0137] When used herein, the term "progression-free survival (PFS)" is used. "The time interval between the date of T-cell infusion and the date of disease progression or death from any cause may be defined. Progression is identified by the investigator's assessment of the patient's response, as defined by the IWG criteria (Cheson et al., J Clin Oncol. 2007;25(5):579-86).

[0138] 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.

[0139] 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.

[0140] 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).

[0141] As used herein, “CAR T cell peak” is defined as the maximum absolute number of CAR+PBMCs / μL in serum achieved after day 0.

[0142] When used in this specification, "CAR T cell peak time" refers to the time from day 0 to CAR This is defined as the number of days until the peak T cell count is reached.

[0143] 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.

[0144] 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).

[0145] As used herein, the "baseline" of cytokines is defined as the last value measured before conditioning chemotherapy.

[0146] When used in this specification, the change in magnification from baseline on day X is:

number

[0147] As used herein, “post-baseline cytokine peak” is defined as the maximum level of serum cytokines achieved from baseline (-5 days) to day 28.

[0148] 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.

[0149] 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.

[0150] In this specification, 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 are defined as follows: Adverse events may be coded using the Drug Regulatory Terminology (MedDRA) version 22.0 and the Common Terminology Criteria for Adverse Events of the National Cancer Institute (NCI). Criteria for Adverse Events (CTCAE) version 4.03 can be used for grading. Cytokine release syndrome (CRS) events can be graded to the syndrome level according to Lee et al. (Lee et al, 2014 Blood. 2014;124(2):188-95). Individual CRS symptoms can be graded according to CTCAE 4.03. Neurological events can be identified using exploratory strategies 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.

[0151] Various aspects of this disclosure are described in more detail in the following subsections. Characterization of serum protein profiles in immunotherapy-treated cancer patients

[0152] In some embodiments, this disclosure provides methods for characterizing the serum proteomics profile of cancer patients prior to immunotherapy and / or preconditioning treatment. 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 oatl-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, 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).

[0153] 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 engineered antigen-specific T-cell receptor (TCR)-positive (+) T cells. In one embodiment, which includes the administration of cells, the immunotherapy is CAR T cell or TCR T cell therapy. In one embodiment, the immunotherapy is anti-CD19 CAR T cell therapy.

[0154] 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.

[0155] In one embodiment, the disclosure provides that baseline (preconditioning) serum levels of specific proteins associated with the IL1 / IL6 pathway positively correlate with and may be biomarkers for Grade 3+ cytokine release syndrome (CRS) after immunotherapy. In one embodiment, the proteins associated with the IL1 / IL6 pathway are selected from IL1A and OSMR. Thus, in one embodiment, the disclosure provides a method for predicting the likelihood that a patient will develop Grade 3+ CRS after immunotherapy based on measurements of baseline serum levels of IL1A and OSMR, where higher serum levels of one or more of IL1A and OSMR indicate a higher likelihood that the patient will develop Grade 3+ CRS after immunotherapy. In one embodiment, this information is used to make immunotherapy decisions, including whether or not to administer immunotherapy, what dose of immunotherapy to administer, what dosing regimen to follow, and / or which drugs to administer to the patient before, after, and / or during immunotherapy, in order to improve management in the patient and / or reduce Grade 3+ CRS.

[0156] In one embodiment, the disclosure provides that baseline (preconditioning) serum levels of certain proteins associated with inflammatory endothelial markers may be positively correlated with both Grade 3+ CRS and Grade 3+ neurological events (NEs) after immunotherapy and may be their biomarkers. In one embodiment, the inflammatory endothelial markers are ACE2, CEACAM1, ICAM2, and ADAM15. In one embodiment, the disclosure provides that baseline serum levels of MET, OSMR, ACE2, ACY1, and BILDIR may be positively correlated with Grade 3+ CRS and NEs after immunotherapy and may be their biomarkers. Therefore, in one embodiment, the disclosure provides that ACE2, CEACAM1, ICAM 2 Based on measurements of baseline serum levels of ADAM15, MET, OSMR, ACY1, and BILDIR, this method provides a way to predict the likelihood of a patient developing grade 3+ CRS and / or grade 3+ NE after immunotherapy, as well as ACE2, CEACAM1, and ICAM 2、The higher the serum level of one or more of ADAM15, MET, OSMR, ACE2, ACY1, and BILDIR, the more likely the patient is to develop grade 3+ CRS and / or grade 3+ NE after immunotherapy. In one embodiment, this information is utilized to make decisions related to immunotherapy, including whether to administer immunotherapy, what dose of immunotherapy to administer, what dosing regimen to follow, and / or what agents to administer to the patient before, after, and / or during administration of immunotherapy, in order to improve management in the patient and / or reduce grade 3+ CRS and / or grade 3+ NE.

[0157] In one embodiment, the present disclosure provides that the baseline serum levels of CCL16, CELA3A, MEGF9, MFAP3, and REG1B on day 0 (i.e., immediately before immunotherapy and after conditioning therapy), and 2 ICAM, LY9, REG3A, SERPINB9, SOD2, ADA2, ITM 2 the serum levels of AREG, BSG CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM on day 0 are positively correlated with grade 3+ CRS and grade 3+ NE after immunotherapy and can be biomarkers thereof. Thus, in one embodiment, the present disclosure provides that one or more of CCL16 2 CELA3A, MEGF9, MFAP3, and REG1B, and / or AREG, BSG CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM 2This method provides a way to predict the likelihood that a patient will develop Grade 3+ CRS and / or Grade 3+ NE after immunotherapy, based on measured Day 0 serum levels of one or more of the following: A, ICOSLG, CGA, CRNN, LY96, AST, and VAMP5, as well as one or more of the following: CCL16, CELA3A, MEGF9, MFAP3, and REG1B, and / or AREG, BSG, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, and ICAM 2 ,LY9,REG3A,SERPINB9,SOD2,ADA2,ITM 2 The higher the day 0 serum level of one or more of A, ICOSLG, CGA, CRNN, LY96, AST, and VAMP5, the more likely the patient is to develop grade 3+ CRS and / or grade 3+ NE after immunotherapy. In one embodiment, this information is used to make immunotherapy-related decisions, including whether or not to administer immunotherapy, 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, in order to improve patient management and / or reduce grade 3+ CRS and / or grade 3+ NE.

[0158] In one embodiment, the disclosure provides that the ratio change between day 0 serum levels and baseline serum levels of GPA33, EPCAM, and CRNN positively correlates with and can be biomarkers for grade 3+ CRS and NE after immunotherapy. In one embodiment, serum levels of one or more of these proteins positively correlate with and can be biomarkers for immune activation-induced stress after immunotherapy, and such markers can be downregulated after lymphocyte depletion. In one embodiment, the conditioning therapy comprises cyclophosphamide and fludarabine. Thus, in one embodiment, the disclosure provides a method for predicting the likelihood that a patient will develop grade 3+ CRS and / or grade 3+ NE after immunotherapy based on the ratio / ratio change between day 0 and baseline serum levels of one or more of the serum levels of GPA33, EPCAM, and CRNN, where a higher ratio between day 0 serum levels and baseline serum levels of one or more of the serum levels of GPA33, EPCAM, and CRNN is the likelihood that the patient will develop grade 3+ CRS and / or grade 3+ NE after immunotherapy. In one embodiment, this information is used to make immunotherapy-related decisions, including whether or not to administer immunotherapy, what dose of immunotherapy to administer, what dosing regimen to follow, and / or which drugs to administer to the patient before, after, and / or during immunotherapy, in order to improve patient management and / or reduce grade 3+ CRS and / or grade 3+ NE. Also, in one embodiment, the Disclosure provides a method for predicting immunotherapy-induced immune activation stress based on the ratio between the day 0 measurement and baseline measurement of one or more serum levels of GPA33, EPCAM, and CRNN, wherein a higher ratio between the day 0 serum level and baseline serum level of one or more of GPA33, EPCAM, and CRNN indicates greater immunotherapy-induced immune activation stress.In one embodiment, this information is used to make immunotherapy-related decisions, including whether or not to administer immunotherapy, what dose of immunotherapy to administer, what administration regimen to follow, and / or which drugs should be administered to the patient before, after, and / or during immunotherapy, in order to reduce immune activation-induced stress after immunotherapy.

[0159] Furthermore, this disclosure provides a method for stratifying patients who are more likely to develop a severe adverse event (e.g., Grade 3+ CRS and / or Grade 3+ NE), and the method This includes administering immunotherapy in combination with one or more agents that reduce immune activation and / or endothelial cell destruction, wherein the combination therapy reduces cytokine induction and / or endothelial cell destruction, and the patient (i) baseline MET, OSMR, ACE2, ACY1, FGF21, BILDIR; day 0 CCL16, CELA3A, MEGF9, MFAP3, REG1B, AREG, BSG CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, OSMR, PAG1, PCDH17, STK11, ACE2, ADAM15, CEACAM1, HLA-DRA, ICAM 2 ,LY9,REG3A,SERPINB9,SOD2,ADA2,ITM 2 The method provides a patient who has high levels of serum markers, which are inferred by measuring the fold change between day 0 and baseline of (ii) one or more protein levels of A, ICOSLG, CGA, CRNN, LY96, AST, and VAMP5, and (ii) the fold change of serum GPA33, EPCAM, and CRNN between day 0 and baseline. In one embodiment, high levels of serum biomarkers are at least 1.5 times higher than the reference level.

[0160] In one embodiment, serum levels for each of the proteins were as follows: baseline 0.56-0.75 (MET, top), 0.62-0.77 (OSMR, top), 0.65-0.98 (ACE2, top), 0.66-1.10 (ACY1, top), 1.58-2.25 (FGF21, bottom), 4.12-5.50 (BILDIR, top); day 0 0.84-1.25 (CCL16, top), 1.37-2.01 (CELA3A, top), 0.26-0.48 (MEGF9, top), 0.33-0.49 (MFAP3, top), 1.49-1.99 (REG1B, top). , 0.88~1.22 (AREG, upper), 0.73~0.96 (BSG, upper) 1.83~2.31 (CKAP4, upper), 3.48~3.91 (CXCL1, upper), 0.09~0.23 (EIF5A, upper), 0.46~0.81 (IL1A, upper), 1.36~1.66 (KIFBP , upper), 1.62~2.13 (KIRREL2, upper), 1.39~1.77 (NUB1, upper), 0.65~0.79 (OSMR, upper), 2.16~2.74 (PAG1, upper), 1.03~1.32 (PCDH17, upper), 1.09~1.35 (STK11, upper), 1.20~1. 65 (ACE2, upper), 1.09~1.31 (ADAM15, upper), 0.69~0.90 (CEACAM1, upper), 1.13~1.41 (HLA-DRA, upper), 1.21~1.43 (ICAM2, upper), 0.11~0.49 (LY9, upper), 2.34~2.88 (REG3A, 2.12~2.30 (SERPINB9, upper), 1.37~1.86 (SOD2, upper), 0.70~0.98 (ADA2, upper), 0.74~1.01 (ITM2A, lower), 0.45~0.56 (ICOSLG, lower), 1.12~1.50 (CGA, upper), -0.48~0. Serum levels are considered high if they are above / below (underlined) 08 (CRNN, sup), 0.54-0.70 (LY96, sup), 42.12-65.80 (AST, sup), and 1.30-1.73 (VAMP5, sup), as well as the divergence change between day 0 and baseline -1.36-(-0.60) (GPA33, sup), -0.82-(-0.39) (EPCAM, sup), and -0.27-0.16 (CRNN, sup). The subject is predicted to have a higher probability of grade 3+ CRS and / or grade 3+ NE and / or is selected for combination therapy.

[0161] 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 multiplier change between day 0 and baseline of blood GPA33, EPCAM, and CRNN, which are triggers for combination therapy, is at least 1.5 The values ​​are 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. In one embodiment, plasma levels of protein biomarkers are high if, when evaluated by those skilled in the art, they fall in the second, third, or fourth quartile of their levels in a representative tumor population. 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".

[0162] In one embodiment, the present disclosure provides a method for treating a subject having a low eosinophil count and / or monocyte count on day 0 by administering one or more agents or treatments that result in a reduction of inflammation.

[0163] In one embodiment, the present disclosure is a method of 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).

[0164] 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.

[0165] In one embodiment, immunotherapy is T-cell therapy. In one embodiment, T-cell therapy is autologous. In one embodiment, T-cell therapy is allogeneic. In some embodiments, T-cell therapy includes adoptive cell therapy. In certain embodiments, 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, 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, immunotherapy is CAR T-cell therapy or TCR T-cell therapy. In one embodiment, immunotherapy is anti-CD19 CAR This is T-cell therapy. Examples of target tumor antigens are listed elsewhere in this specification. Examples of cancers that can be treated by the methods disclosed herein are also shown elsewhere in this specification.

[0166] In one embodiment, an agent(s) administered in combination with immunotherapy to reduce immune activation and / or endothelial cell destruction, wherein the combination therapy reduces cytokine induction and / or reduces endothelial cell destruction, and 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 is performed The drugs include grolimab (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) lenzilumab; namilumab (AMG203); GSK3196165 / MOR103 / ocilimab (GSK / MorphoSys); KB00 (ii) 2 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022 / demicirmab (Morphotek); or any of these biosimilars; E21R; and GM-CSF inhibitors selected from small molecules; (ii) RG7155, PD-0360324, MCS110 / lacnotuzumab, or any of these biosimilar versions; and CSF1 inhibitors selected from small molecules; and / or (iii) GM-CSFR inhibitors, as well as maprilimumab (formerly CAM-3001; MedImmune, Inc.); kabilizumab (Five Emactuzumab, also known as Prime Therapeutics; 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.

[0167] 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 the CAR-T peak proliferation is days 14-28 after infusion. This is a period of days. 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 after peak proliferation.

[0168] 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 drug is checkpoint The inhibitor is selected from (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)).

[0169] 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.

[0170] 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 involves an immunomodulatory imide drug (IMID) / cerebron modulator (e.g., lenalidomide, pomalidomide, This includes administering bridging therapy with iverdomede and apremilast. In one embodiment, optimization includes administering bridging therapy by local radiation.

[0171] 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.

[0172] 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.

[0173] 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).

[0174] 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.

[0175] 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 / mm³) 2 The method includes (within the range of) and / or high TME myeloid inflammation levels (within the range of 27 to 2000). In one embodiment, the method comprises manipulating CAR T cells to express a gamma chain receptor cytokine. In one embodiment, the gamma chain receptor cytokine is expressed under a constitutive or inductive promoter. In one embodiment, the method comprises growing T cells in the presence of a gamma chain cytokine such as IL-15. Clinical Outcomes

[0176] In some embodiments, the clinical outcome is complete response. In some embodiments, the clinical outcome is sustained response. In some embodiments, the clinical outcome is complete response. In some embodiments, the clinical outcome is no response. In some embodiments, the clinical outcome is partial response. In some embodiments, the clinical outcome is objective response. In some embodiments, the clinical outcome is survival. In some embodiments, the clinical outcome is relapse.

[0177] In some embodiments, objective response (OR) is determined according to the revised IWG treatment response criteria for malignant lymphoma (Cheson, 2007) and the IWG treatment response criteria for malignant lymphoma (Cheson et al. Journal of Clinical Oncology 32, no. 27 (September 2014) 3059-3067). The duration of response is evaluated. Progression-free survival (PFS) is evaluated by the investigator according to the Lugano classification response criteria.

[0178] In some embodiments, part of the clinical outcome is the evaluation of adverse events. In this regard, CRS grading was 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 patients 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.

[0179] 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. The method includes monitoring the patient for signs or symptoms of neurotoxicity for four weeks after injection.

[0180] 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.

[0181] The clinical outcome of CAR T cell therapy depends 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 week, 2 weeks, 3 weeks, or 4 weeks after administration of engineered CAR T cells. The 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 manipulated CAR T cells. In some embodiments, the 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 manipulated CAR T cells.

[0182] 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%. These ranges include percentages such as 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

[0183] 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. CARs can be programmed so that a single receptor both recognizes a specific antigen and, upon binding to that antigen, activates 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 co-stimulatory (signaling) domains to enhance their potency. (U.S. Patents 7,741,465 and 6,319,494, and Krause et al. and Finney et al., Song) et al.,Blood 119:696-706(2012);Kalos et al. al., Sci. Transl. Med. 3:95 (2011), Porter et al. See al., N.Engl.J.Med.365:725-33(2011) and Gross et al., Annu.Rev.Pharmacol.Toxicol.56:59-83(2016).

[0184] 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.

[0185] 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.

[0186] 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. The transmembrane regions particularly useful in this disclosure are 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, CD3ζ, 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, 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( It may be derived from 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, Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof (for example, may include at least a transmembrane domain).

[0187] Optionally, short linkers can form connections between one or more of the extracellular, transmembrane, and intracellular domains of a CAR. An example of such short linkers is described in International Publication 2019 / 060695, which is incorporated in its entirety by reference.

[0188] 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 contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0189] 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 the addition of a small molecule, the spacer domain is a chemoinducible dimerizing factor. This may include spacers. In some embodiments, spacers are not used.

[0190] The intracellular (signaling) domain of the manipulated T cell described herein may result in signaling to an activation domain, which then activates at least one of the normal effector functions of the immune cell. The effector function of the T cell may be, for example, cytolytic activity or helper activity, which may include cytokine secretion.

[0191] 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 This includes the following molecules: S1 molecule, NKG2C, 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

[0192] 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, e.g., 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. scFvs retain the ability of a parent antibody to specifically interact with the target antigen. scFvs are useful in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain together with other CAR components (ibid.). See also Krause et al., J.Exp.Med., Volume 188, No.4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It will be understood that antigen-binding molecules are usually contained in the extracellular portion of a 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.

[0193] In some embodiments, the polynucleotide has a (shortened) hinge domain and a target antibody The CAR or TCR encodes an antigen-binding molecule that specifically binds to the target. 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), ELF2 M, 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

[0194] In one embodiment, the 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, the T cells are tumor-infiltrating lymphocytes (TILs), manipulated autologous T cells (eACT®), allogeneic T cells, heterologous T cells, or any combination thereof. In some embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient with cancer or a tumor. In some embodiments, the donor subject is a human patient who does not have cancer or a tumor.

[0195] 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 infection sites, ascites, pleural fluid, splenic tissue, 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 obtained from plasma. The cells are washed to remove impurities and placed in a suitable buffer or culture medium for further processing. In some embodiments, the cells are washed with PBS. As understood, the washing step may be performed by using, for example, a semi-automatic flow-through centrifuge, e.g., a Cobe® 2991 cell processing device, a Baxter CytoMate®, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other salines with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. 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.

[0196] 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.

[0197] 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 the 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.

[0198] 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.

[0199] 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 chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding CARs), and then activated and / or proliferated in vitro. Methods for activating and proliferating T cells are known in the Art, for example, U.S. Patent No. 6,900 This is described in publications 5,874, 6,867,041, and 6,797,514, and International Publication No. 2012 / 079000, the contents of which are incorporated herein by reference in their entirety. Generally, such methods involve contacting 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 by methods described in U.S. Patent No. 6,040,177 and No. 5,827,642 and International Publication No. 2012 / 129514 (the contents thereof are incorporated herein by reference in their entirety).

[0200] 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 an excipient. A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0201] 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.

[0202] 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 6 Individual cells / kg, approximately 3×10 6Individual 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.

[0203] 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 Individual operation This is the period until the maximum dose of surviving T cells is reached.

[0204] 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.

[0205] This application also provides doses and administrations of cells prepared by the method of this application, for example, an infusion bag for CD19-directed genetically modified autologous T cell immunotherapy contains approximately 68 mL of chimeric antigen receptor (CAR)-positive T cell suspension for infusion. In some embodiments, 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 contains 1 × 10¹⁶ 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 ~Approx. 2×10 6 These may be CAR-positive surviving T cells, up to 2 × 10⁶ 8 These could be CAR-positive surviving T cells.

[0206] 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.

[0207] In some embodiments, the CD19-directed T-cell immunotherapy is KTE-X19, which is prepared as described elsewhere in this application. In one embodiment, KTE-X19 may be used to treat MCL, ALL, CLL, SLL, and any other B-cell malignancies. In some embodiments, the CD19-directed genetically modified autologous T-cell immunotherapy is Axi-cel® (YESCARTA®, axicaptagensilolucel), which is prepared by one of the methods of this application. The amount of CAR T cells, dosing regimens, administration methods, subjects, and cancers within the scope of these methods are described elsewhere in this application, either alone or in combination with other chemotherapeutic agents, with or without pretreatment, for any of the patients described elsewhere in this application. Conditioning agent

[0208] In some embodiments, the subject is administered a conditioning agent before immunotherapy. In some embodiments, conditioning is performed by radiation therapy. In one embodiment, the conditioning therapy is lymphocyte apheresis chemotherapy.

[0209] In one embodiment, the conditioning therapy includes an alkylating agent selected from the group consisting of melphalan, chlorambucil, cyclophosphamide, mechloretamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozosin, alkyl sulfonates, busulfan, thiotepa or analogues thereof, and any combination thereof, azathioprine, 6-mercaptopurine, mercaptopurine, and The present invention comprises purine analogs selected from the group consisting of oprin, thioguanine, fludarabine, pentostatin, cladribine, and any combination thereof, and / or platinum-based preconditioning agents selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomid, temozolomid, dacarbazine, temozolomid, and any combination thereof.

[0210] Cyclophosphamide (ENDOXAN®, CYTOXAN®, PROCYTOX®, NEOSAR®, REVIMMUNE®, CYCLOBLASTIN®) is a nitrogen mustard-derived alkylating agent with potent immunosuppressive activity. Cyclophosphamide acts as an antineoplastic agent and is used to treat various types of cancer, including lymphoma, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian cancer, eye cancer, and breast cancer, as well as autoimmune disorders.

[0211] When administered to patients, cyclophosphamide is converted to acrolein and phosphoramide in the liver. Simultaneously, these metabolites crosslink DNA in both resting and dividing cells by adding alkyl groups to the guanine bases of DNA at the nitrogen atom at position 7 of the imidazole ring. As a result, DNA replication is inhibited, leading to cell death.

[0212] In another embodiment, one or more preconditioning agents may include platinum-based chemotherapeutic agents. In a particular embodiment, 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 analogues or functional derivatives thereof, and any combination thereof.

[0213] In another embodiment, one or more preconditioning agents may include purine analogs. In a particular embodiment, the purine analogs are 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 preconditioning agents include fludarabine.

[0214] Fludarabine phosphate (FLUDARA®) is a synthetic purine nucleoside that differs from physiological nucleosides in that its sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine acts as a purine antagonist and is used to treat various types of hematological malignancies, including various lymphomas and leukemias.

[0215] Upon administration to a patient, fludarabine is rapidly dephosphorylated to 2-fluoro-ara-A, and then phosphorylated intracellularly by deoxycytidine kinase to activate triphosphate. It is converted to the acid 2-fluoro-ara-ATP. This metabolite then interferes with DNA replication and thus inhibits DNA synthesis, likely by inhibiting DNA polymerase α, ribonucleotide reductase, and DNA primase. As a result, fludarabine administration leads to increased cell death in dividing cells.

[0216] In some embodiments, one or more preconditioning agents may include cyclophosphamide and purine analogs. The 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.

[0217] 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 / m 2 / day, or approximately 600 mg / m² 2 / day~about 2000mg / m 2 In other embodiments, the dose of cyclophosphamide is approximately 350 mg / m². 2 / day~about 1500mg / m 2 / day, about 350mg / m 2 / day~about 1000mg / m 2 / day, about 400 mg / m 2 / day to about 900 mg / m 2 / day, about 450 mg / m 2 / day to about 800 mg / m 2 / day, about 450 mg / m 2 / day to about 700 mg / m 2 / day, about 500 mg / m 2 / day to about 600 mg / m 2 / day, or about 300 mg / m 2 / day to about 500 mg / m 2 / day. In another embodiment, the dose of cyclophosphamide is about 350 mg / m 2 / day, about 400 mg / m 2 / day, about 450 mg / m 2 / day, about 500 mg / m 2 / day, about 550 mg / m 2 / day, about 600 mg / 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.

[0218] In other embodiments, the first dose of cyclophosphamide (which also applies to the repeated dose) is about 200 mg / m 2 / day to about 3000 mg / m 2 / day. In another embodiment, the first dose of cyclophosphamide is higher than 200 mg / m 2 / day and lower than 3000 mg / m 2 / day. In other embodiments, the dose of cyclophosphamide is about 200 mg / m 2 / day to about 3000 mg / m 2 / day, about 300 mg / m 2 / day to about 3000 mg / m 2 / day, about 400 mg / m 2 / day to about 3000 mg / m 2 / day, about 500 mg / m 2 / day to about 3000 mg / m 2 / day, about 600 mg / 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, approximately 500mg / m 2 / Daily ~ Approximately 2700mg / m 2 / day, approximately 600mg / m 2 / Daily ~ Approximately 2600mg / m 2 / day, approximately 700mg / m 2 / Daily ~ Approximately 2500mg / m 2 / day, approximately 800mg / m2 / 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, approximately 1100mg / m 2 / day ~ approx. 2250mg / m 2 / day, or approximately 1110 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

[0219] 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 / m 2 / day, about 80mg / m 2 / day, about 85mg / m 2 / day, approximately 90mg / m 2 / day, approximately 95mg / m 2 / day, about 100mg / m 2 / day, about 200mg / m 2 / day, or approximately 300 mg / m²2 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, approximately 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. 2This 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

[0220] In some embodiments, the dose of cyclophosphamide is 100 mg / m². 2 / day (or 110mg / m²) 2 / day, 120mg / m 2 / day, 130mg / m 2 / day, or 140 mg / m 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0221] In some embodiments, the dose of cyclophosphamide is 150 mg / m². 2 / day (or 160mg / m²) 2 / day, 170mg / m 2 / day, 180mg / m 2 / day, or 190mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0222] In some embodiments, the dose of cyclophosphamide is approximately 200 mg / m². 2 / day (or 210 mg / m²) 2 / day, 220mg / m 2 / day, 230mg / m 2 / day, or 240mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0223] In some embodiments, the dose of cyclophosphamide is 250 mg / m². 2 / day (or 260mg / m²) 2 / day, 270mg / m 2 / day, 280mg / m 2 / day, or 290mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0224] In some embodiments, the dose of cyclophosphamide is 300 mg / m². 2 / day (or 310mg / m²) 2 / day, 320mg / m 2 / day, 330mg / m 2 / day, or 340mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0225] In some embodiments, the dose of cyclophosphamide is 350 mg / m². 2 / day (or 360mg / m²) 2 / day, 370mg / m 2 / day, 380mg / m 2 / day, or 390mg / m² 2The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0226] In some embodiments, the dose of cyclophosphamide is 400 mg / m². 2 / day (or 410mg / m²) 2 / day, 420mg / m 2 / day, 430mg / m 2 / day, or 440mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0227] In some embodiments, the dose of cyclophosphamide is 450 mg / m². 2 / day (or 460mg / m²)2 / day, 470mg / m 2 / day, 480mg / m 2 / day, or 490mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0228] In some embodiments, the dose of cyclophosphamide is 500 mg / m². 2 / day (or 510mg / m²) 2 / day, 520mg / m 2 / day, 530mg / m 2 / day, or 540mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0229] In some embodiments, the dose of cyclophosphamide is 550 mg / m². 2 / day (or 560mg / m²) 2 / day, 570mg / m 2 / day, 580mg / m 2 / day, or 590mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0230] In some embodiments, the dose of cyclophosphamide is 600 mg / m². 2 / day (or 610mg / m²) 2 / day, 620mg / m 2 / day, 630mg / m 2 / day, or 640mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m2 / day, or 75mg / m² 2 / day

[0231] In some embodiments, the dose of cyclophosphamide is 650 mg / m². 2 / day (or 660mg / m²) 2 / day, 670mg / m 2 / day, 680mg / m 2 / day, or 690mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0232] In some embodiments, the dose of cyclophosphamide is 700 mg / m². 2 / day (or 710mg / m²) 2 / day, 720mg / m 2 / day, 730mg / m 2 / day, or 740mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / 75 mg / m² per day, or 75 mg / m² 2 / day

[0233] In some embodiments, the dose of cyclophosphamide is 750 mg / m². 2 / day (or 760mg / m²) 2 / day, 770mg / m 2 / day, 780mg / m 2 / day, or 790mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0234] In some embodiments, the dose of cyclophosphamide is 800 mg / m². 2 / day (or 810 mg / m²) 2 / day, 820mg / m 2 / day, 830mg / m 2 / day, or 840 mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0235] In some embodiments, the dose of cyclophosphamide is 850 mg / m². 2 / day (or 860mg / m²) 2 / day, 870mg / m 2 / day, 880mg / m 2 / day, or 890mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0236] In some embodiments, the dose of cyclophosphamide is 900 mg / m². 2 / day (or 910mg / m²) 2 / day, 920mg / m 2 / day, 930mg / m 2 / day, or 940mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0237] In some embodiments, the dose of cyclophosphamide is 950 mg / m². 2 / day (or 960mg / m²) 2 / day, 970mg / m 2 / day, 980mg / m 2 / day, or 990mg / m² 2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0238] In some embodiments, the dose of cyclophosphamide is 1000 mg / m². 2 / day (or 1010 mg / m²) 2 / day, 1020mg / m 2 / day, 1030mg / m 2 / day, or 1040 mg / m²2 The dosage is 5 mg / m² (per day), and the dose of fludarabine is 5 mg / m². 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75mg / m² 2 / day

[0239] In other embodiments, the dose of cyclophosphamide is 100 mg / m². 2 / day~650mg / m 2 The daily dose is 10 mg / m² of fludarabine. 2 / day~50mg / m 2 In other embodiments, the dose of cyclophosphamide is 150 mg / m². 2 / day~600mg / m 2 The daily dose is 20 mg / m² of fludarabine. 2 / day~50mg / m 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m². 2 / day~550mg / m 2 The daily dose is 20 mg / m² of fludarabine. 2 / day~40mg / m 2 In other embodiments, the dose of cyclophosphamide is 250 mg / m². 2 / day~550mg / m 2 The daily dose is 15 mg / m² of fludarabine. 2 / day~45mg / m 2 / day

[0240] In a particular embodiment, the dose of cyclophosphamide is 1000 mg / m².2 The daily dose is 60 mg / m² of fludarabine. 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, 75mg / m 2 / day, 80mg / m 2 / day, 85mg / m 2 / day, 90mg / m 2 / day, 95mg / m 2 / day, 100mg / m 2 / day, 105mg / m 2 / day, 110mg / m 2 / day, 115mg / m 2 / day, 120mg / m 2 / day, 125mg / m 2 / day, 130mg / m 2 / day, 135mg / m 2 / day, 140mg / m 2 / day, 145mg / m 2 / day, 150mg / m 2 / day, 155mg / m 2 / day, 160mg / m 2 / day, 165mg / m 2 / day, 170mg / m 2 / day, 175mg / m 2 / day, 180mg / m 2 / day, 185mg / m 2 / day, 190mg / m 2 / day, 195mg / m 2 / day, 200mg / m 2 / day, 205mg / m 2 / day, 210mg / m 2 / day, 215mg / m 2 / day, 220mg / m 2 / day, 225mg / m 2 / day, 230mg / m 2 / day, 235mg / m 2 / day, 240mg / m 2 / day, 245mg / m 2 / day, or 250 mg / m² 2 / day

[0241] In one embodiment, the dose of cyclophosphamide is approximately 500 mg / m².2 The daily dose of fludarabine is approximately 60 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 300 mg / m². 2 The daily dose of fludarabine is approximately 30 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 200 mg / m². 2 The daily dose of fludarabine is approximately 20 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 200 mg / m². 2 The daily dose of fludarabine is approximately 30 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 500 mg / m². 2 The daily dose of fludarabine is approximately 30 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 300 mg / m². 2 The daily dose of fludarabine is approximately 60 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 500 mg / m². 2 The daily dose of fludarabine is approximately 60 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 1110 mg / m². 2 The daily dose of fludarabine is approximately 25 mg / m². 2 This is / day. In another embodiment, the dose of cyclophosphamide is approximately 2220 mg / m². 2 The daily dose of fludarabine is approximately 25 mg / m². 2 In another embodiment, the dose of cyclophosphamide is approximately 500 mg / m². 2 The daily dose of fludarabine is approximately 30 mg / m². 2 / day

[0242] 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.

[0243] 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 can be decreased, and the dose of cyclophosphamide can be decreased while the dose of fludarabine can be decreased. The dose of darabine can be 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 of fludarabine. 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 of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 500 mg / m². 2 The daily dose is 60 mg / m² of fludarabine. 2In 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 of fludarabine. 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m². 2 The daily dose is 60 mg / m² of fludarabine. 2 / day

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, one or more preconditioning agents are administered daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. It can be administered daily. In one particular embodiment, one or more preconditioning agents are administered daily for about three days.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] One or more preconditioning agents may be administered by any route, including intravenous (IV) or oral. In some embodiments, one or more preconditioning agents, for example, cyclophosphamide, are administered IV over approximately 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, and 120 minutes. In some embodiments, one or more preconditioning agents, for example, fludarabine, are administered IV over approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, and 120 minutes.

[0252] In another embodiment, cyclophosphamide 500 mg / m² 2 and fludarabine 30 mg / m² 2 The lymphocyte apheresis chemotherapy regimen is administered intravenously on days 5, 4, and 3 prior to the infusion of CAR T cells into the subject. In some embodiments, the high-intensity or low-intensity lymphocyte apheresis regimen has a Cy / Flu system (the high-intensity regimen contains 60 mg / kg of cyclophosphamide, and the low-intensity regimen contains 1500 mg / m²). 2 (The following or including cyclophosphamide in a total dose of 30 mg / kg). In some embodiments, the conditioning regimen is 30 mg / m² of fludarabine daily for 3 days. 2 and cyclophosphamide 300 mg / m² 2 This consists of (for example, the final day is -7 to -2 days before CAR T cell infusion). In some embodiments, the conditioning regimen includes fludarabine (30 mg / m²) on -5, -4, and -3 days before CAR T cell infusion. 2 ( / day) and cyclophosphamide (300 mg / m²) 2 ) consists of. cancer

[0253] 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.

[0254] 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 blood cancer. In some embodiments, cancer is a cancer of the white blood cells. In other embodiments, cancer is a cancer of the plasma cells. In some embodiments, cancer is leukemia, lymphoma, or myeloma.In some embodiments, cancer includes 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 myeloid leukemia (CML), chronic or acute granulomatous disease, and chronic myeloid leukemia. Furthermore, 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, spinal cord 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 (Cr These include ow-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.

[0255] 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).

[0256] 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.

[0257] In some embodiments, the cancer is advanced-stage low-grade non-Hodgkin lymphoma (iNHL), which includes follicular lymphoma (FL) and marginal zone lymphoma (MZL). In some embodiments, the patient is treated with a combination of an anti-CD20 monoclonal antibody and an alkylating agent. In rare cases, patients developed relapsed / treatment-resistant disease after two or more prior treatments. In some embodiments, the patient may have been administered a PI3K inhibitor. In some embodiments, the patient may have also undergone autologous stem cell transplantation. In some embodiments, the patient underwent 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., axicapbutagen silol-ucel) at a targeted 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 shows progress after demonstrating an effect at an evaluation 3 months after the initial administration, the patient may be re-treated with CAR T cell therapy (e.g., axicapbutagen silol-ucel). In some embodiments, the patient may receive bridging therapy. Examples of bridging therapy are posted 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.

[0258] 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 6 Individual cells, at least about 10 7 Individual cells, at least about 108 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 the ZUMA-5 clinical trial (NCT03105336).

[0259] 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, LBCL is MYC and BCL2 translocation and / or high-risk / high-grade LBCL with BCL6 translocation or DLBCL exhibiting an IPI score of 3 or higher at any point prior to enrollment. 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, CAR T-cell therapy is administered first. In some embodiments, a regimen containing an anti-CD20 monoclonal antibody / anthracycline is administered first. In some embodiments, treatments are administered at intervals such as 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, or 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 an ECOG PS of 0-1. In some embodiments, the conditioning therapy includes fludarabine 30 mg / m² on days -5, -4, and -3. 2 IV infusion and cyclophosphamide 500 mg / m² 2 This includes IV injection of [the substance]. Other exemplary beneficial preconditioning treatment regimens are described in U.S. Provisional Patent Applications No. 62 / 262,143 and No. 62 / 167,750 and U.S. Patents No. 9,855,298 and No. 10,322,146, which are incorporated herein by reference in their entirety. These include, for example, cyclophosphamide (200 mg / m²) in specified beneficial doses. 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 axicaptagensilolucel or YESCARTA®. In some embodiments, the CAR T-cell therapy includes TECARTUS®-brexcabutagen otreucel / KTE-X19 or KYMRIAH®-(tisagen lecleucel), etc., idekabutagen otreucel / bb2121. In some embodiments, the therapeutic method is one used in any of the ZUMA-1 to ZUMA-19, KITE-585, KITE-222, KITE-037, KITE-363, KITE-439, or KITE-718 clinical trials, which have been well described in the Art.

[0260] In another embodiment, the Disclosure provides a method for treating cancer in a subject requiring treatment, 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, 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.

[0261] 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 axicaptagen silol-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).

[0262] 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 the first-line treatment, and individuals intolerant to the 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 therapy following complete remission to the first-line treatment. In some embodiments, patients selected for second-line axicapbutagen silol-ucel treatment receive fludarabine 30 mg / m² on days -5, -4, and -3. 2 IV and cyclophosphamide 500 mg / m² 2 A conditioning therapy including IV is provided. In some embodiments, axicaptagen silolucel therapy is used as a second-line treatment. Combination therapy

[0263] 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; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; ethyleneimines and methylamelamine; nitro Mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kakutinomycin, calicarea Mycin, 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, for example. Methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamipurine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitio Stan, testolactone; anti-adrenaline, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid, acegraton; aldofamide glycoside; aminolevulinic acid; amsacrine; bestrabusil, bisanthren; edatrexate; defofamine; demecoltin; diazion; elformitin, eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamine; mitogluazone; 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 docetaxel (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.; Examples include Targretin® (bexarotene), Panretin® (alitretinoin); ONTAK® (denileukin difutytox); 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 may also be administered, including CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, R-CHOP (CHOP plus rituximab), and G-CHOP (CHOP plus obinutuzumab).

[0264] 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 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 administering two or more chemotherapeutic agents.

[0265] 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).

[0266] 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®), trastuzumab (HERCEPTIN®), trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), and cetuximab (ERBITUX®). ), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, s Nitinib, sorafenib, toceranib, restoltinib, axitinib, cejiranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib In addition to ponatinib, radotinib, bosutinib, restortinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, 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, antithymocyte globulin, rengilumab, and mabrilimumab are also mentioned.

[0267] 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 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); any of these biosimilar versions; and selected from small molecules.

[0268] 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.

[0269] In some embodiments, the compositions described herein are administered together with cytokines (before, after, or concurrently with T cell administration). Examples of cytokines include: These include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, and glycoprotein hormones, such as follicle-stimulating hormone (FOLLOWED). Thyroid-stimulating hormone (FSH) TSH, luteinizing hormone (LH), hepatic growth factor (HGF) Fibroblast growth factor (FGF); prolactin, placental lactogen, murelan inhibitor; mouse gonadotropin-related peptide; inhibin, activin, vascular endothelial growth factor, 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 factors, interferon, e.g., These include cytokines-alpha, beta, and gamma; colony-stimulating factors (CSFs), e.g., macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), e.g., IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15; tumor necrosis factor, e.g., TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit 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.

[0270] 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, 1 hour or less, or 30 minutes or less.In some embodiments, the administration of cells and additional therapeutic agents occurs between 0 hours or approximately 0 hours and 48 hours or approximately 48 hours, between 0 hours or approximately 0 hours and 36 hours or approximately 36 hours, between 0 hours or approximately 0 hours and 24 hours or approximately 24 hours, between 0 hours or approximately 0 hours and 12 hours or approximately 12 hours, between 0 hours or approximately 0 hours and 6 hours or approximately 6 hours, between 0 hours or approximately 0 hours and 2 hours or approximately 2 hours, between 0 hours or approximately 0 hours and 1 hour or approximately 1 hour, between 0 hours or approximately 0 hours and 30 minutes or approximately 30 minutes, 30 Between 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, 1 hour or Between approximately 1 hour and 36 hours or between approximately 36 hours, between 1 hour or between approximately 1 hour and 24 hours or between approximately 24 hours, between 1 hour or between approximately 1 hour and 12 hours or between approximately 12 hours, between 1 hour or between approximately 1 hour and 6 hours or between approximately 6 hours, between 1 hour or between approximately 1 hour and 4 hours or between approximately 4 hours, between 1 hour or between approximately 1 hour and 2 hours or between approximately 2 hours, between 2 hours or between approximately 2 hours and 48 hours or between approximately 48 hours, between 2 hours or between approximately 2 hours and 36 hours or between approximately 36 hours, between 2 hours or between approximately 2 hours and 24 hours or between approximately 24 hours, between 2 hours or approximately 2 Between 12 hours or approximately 12 hours, between 2 hours or approximately 2 hours and 6 hours or approximately 6 hours, between 2 hours or approximately 2 hours and 4 hours or approximately 4 hours, between 4 hours or approximately 4 hours and 48 hours or approximately 48 hours, between 4 hours or approximately 4 hours and 36 hours or approximately 36 hours, 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, 6 hours or approximately 6 hours. The procedure is performed over a period of up to 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, the cells and additional therapeutic agents are administered simultaneously.

[0271] 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.

[0272] 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.

[0273] In some embodiments, the drug(s) may be administered by injection, for example, intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, choroidal injection, anterior chamber injection, subconjunctival injection, subconjunctival injection, It is administered by sub-Tenon injection, retrobulbar injection, peribulbar injection, or delivery near the posterior sclera. In some embodiments, it is administered parenterally, intrapulmonaryly, and intranasally, or intra-lesionally if local treatment is preferred. Parenteral administration methods include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0274] 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, MK-2206. In one embodiment, the mTOR inhibitor is selected from everolimus, sirolimus, temsirolimus, and ridaflorimus. In some embodiments, PI3K / The mTOR dual inhibitor is selected from BEZ235, XL765, and GDC-0980. 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).

[0275] 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.

[0276] 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).

[0277] [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

[0278] In some embodiments, the administration of immunotherapy (e.g., chimeric receptor T-cell immunotherapy) is performed at an accredited medical institution.

[0279] 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 severe adverse reactions

[0280] In some embodiments, the method includes the management of adverse reactions in any subject. In some embodiments, the adverse reactions are selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, severe infections, cytopenia, and hypogammaglobulinemia.

[0281] In some embodiments, this disclosure provides methods for preventing the development of adverse reactions or reducing the severity of adverse reactions based on levels of numerous biomarkers in the serum of a subject receiving immunotherapy. In some embodiments, cell therapy is administered with one or more agents that prevent, delay the development 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 a subject who may be prone to disease but has not yet been diagnosed with the disease.

[0282] 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.

[0283] 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.

[0284] 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).

[0285] In some embodiments, adverse events / reactions may be selected from one or more of the following (Table 2).

[0286] [Table 2] Cytokine release syndrome (CRS)

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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)

[0291] 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.

[0292] In some embodiments, the subject experiences Grade 3+ NT. In some embodiments, this includes brain damage, tremors, confusional states, 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

[0293] 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.

[0294] 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 method is outlined in Figure 1. 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 provides early steroid therapy in cohort 4. The disclosure presents that Lloyd's intervention 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 steroid use in cohort 4 resulted in a median cumulative cortisone equivalent dose that was approximately 15% of that in cohorts 1+2, suggesting that early steroid use may enable 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 presents that tocilizumab is initiated for the management of all cases of grade 1 CRS if no improvement is seen after 3 days for all grade ≥ 2 neurological events. In one embodiment, the disclosure provides a method for reducing total steroid exposure in patients undergoing adverse event management after CAR T cell administration, the method comprising initiating corticosteroid therapy for the management of all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 1 neurological events, and / or initiating tocilizumab for all cases of Grade 1 CRS if no improvement is seen after 3 days for all Grade ≥ 2 neurological events. In one embodiment, the corticosteroid and tocilizumab are administered in a regimen selected from the regimens 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.

[0295] 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 if a grade ≥ 2 neurotoxicity develops, levetiracetam is administered. 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.

[0296] 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.

[0297] In one embodiment, this 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. Therefore This disclosure provides a method for managing adverse events in CAR T-cell therapy as shown in Figure 1. In one embodiment, the patient is administered levetiracetam starting on day 0 (750 mg twice daily, orally or intravenously). Upon the onset of a grade 2 or higher neurological event, the dose of levetiracetam is increased to 1000 mg twice daily. If the patient does not exhibit any grade 2 or higher neurological events, levetiracetam is tapered and discontinued as clinically necessary. On day 2, the patient is also administered tocilizumab (8 mg / kg IV over 1 hour [not exceeding 800 mg]). Further tocilizumab (± corticosteroids) may be recommended upon the onset of grade 2 CRS in patients with comorbidities or in elderly patients, or otherwise in cases of grade 3 or higher CRS. Tocilizumab is initiated in patients exhibiting a Grade 2 or higher neurological event. Corticosteroids are added in patients with comorbidities, elderly patients, or in patients experiencing a Grade 3 or higher neurological event that worsens despite tocilizumab use.

[0298] 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.

[0299] In one embodiment, the disclosure shows 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.

[0300] 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.

[0301] 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.

[0302] 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, TNF alpha, TNFR1, e.g., TL-6 receptor (IL-6R), IL-2 receptor (IL-2R / CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-beta receptor (TGF-βI, II, or III), IFN-gamma receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF alpha receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra / IL-1RP) or IL-10 receptor (IL-10R), IL-1 and IL-1R alpha / IL-1 beta. In some embodiments, the drugs are siltuximab, sarilumab, olokizumab (CDP6038), elcilimomab, ALD518 / BMS-945429, silkumab (CNTO 136), CPSI-2634, and ARGX. The agents include 109, FE301, or FM101. In some embodiments, the agent 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 antagonists thereof), such as 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 cell administration by one of the methods and dosages described elsewhere herein.

[0303] 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 the administration of cells by one of the methods and doses described elsewhere in this specification.

[0304] 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, patients exhibiting severe CRS should be considered to undergo 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.

[0305] In some embodiments, adverse events are managed according to the following protocol (Protocol A / Table 1).

[0306] [Table 3] (a)Lee DW et al.(2014).Current concepts in the diagnosis and management of cytokine release syndrome.Blood.2014 Jul 10;124(2):188-195. (b) Refer to Protocol B for the management of neurotoxicity. (c) For further details, see ACEMTRA® (tocilizumab) prescribing information, https: / / www.gene.com / download / pdf / actemra_prescribing.pdf (last accessed October 18, 2017). The initial US approval was indicated in 2010. (d) Alternative therapies include, but are not limited to, anakinra, siltuximab, ruxolitinib, cyclophosphamide, IVIG, and ATG. Neurotoxicity

[0307] 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.

[0308] In some embodiments, the NE is managed according to the following protocol (Protocol B / Table 4).

[0309] [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

[0310] 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.

[0311] 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.

[0312] 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;

[0313] 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.

[0314] 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.

[0315] 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.

[0316] In one embodiment, the protocol for treating adverse events includes protocol C as follows (Table 5):

[0317] [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 an adverse event, CRS indicates cytokine release syndrome, IV indicates intravenous administration, N / A indicates not applicable, and NE indicates a neurological event.

[0318] 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, ha Lucinonide, 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, valerian Hydrocortisone acid, roteprednol ethanolate, mazipredone, medrisone, meprednisone, methylprednisolone (methylprednisolone acetone, 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 phosphate Examples include, but are not limited to, renesylates, 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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

[0325] In some embodiments, patients treated with CAR T cells (e.g., CD19-directed) or other genetically modified autologous T cell immunotherapy may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies. Methods and compositions for producing products for increased clinical efficacy and / or reduced toxicity

[0326] In one embodiment, the present disclosure provides a method for producing an immunotherapy product having improved clinical efficacy and / or reduced toxicity for use in a patient according to the predicted toxicity grade (CRS / NE). In some embodiments, the immunotherapy product comprises blood cells. In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to place the cells into a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some embodiments, the washing step is achieved using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter) according to the manufacturer's instructions for use. In some embodiments, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions for use. In some embodiments, after washing, the cells are resuspended in various biocompatible buffers, such as PBS that does not contain Ca++Mg++. In a particular embodiment, components of the blood cell sample are removed, and the cells are directly resuspended in the culture medium.

[0327] In some embodiments, the method includes density-based cell separation, such as erythrocyte lysis and centrifugation by Percoll or Ficoll density gradient method to prepare leukocytes from peripheral blood. In some embodiments, the method includes leukocyte apheresis.

[0328] In some embodiments, at least part of the selection step includes incubation with a cell selection reagent. For example, as part of a selection method, incubation with a selection reagent or a plurality of selection reagents may be performed using one or more selection reagents for the selection of one or more different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, either intracellularly or on a cellular basis. In some embodiments, any known method using a selection reagent or a plurality of selection reagents for separation based on such markers may be used. In some embodiments, the selection reagent or a plurality of selection reagents results in separation, which is separation based on affinity or immunoaffinity. For example, selection in some embodiments includes incubation with a reagent for the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds to such markers, usually followed by a washing step and separation of cells bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner.

[0329] In some embodiments of such a process, a certain volume of cells is mixed with a certain amount of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in a favorable energetic interaction between the isolated cells and a molecule that specifically binds to a marker on the cell, e.g., an antibody or other binding partner on a solid surface, e.g., particles. In some embodiments, the method is performed using particles such as beads, e.g., magnetic beads, coated with a cell marker-specific selection agent (e.g., an antibody). The particles (e.g., beads) are shaken or mixed in a container such as a tube or bag, providing a constant environment that helps facilitate the energetically favorable interaction. Cells can be incubated or mixed with particles (e.g., beads) at a cell density to particle ratio. In other cases, the method involves all or part of the selection of cells, which is performed, for example, in the internal cavity of a chamber under centrifugal rotation. In some embodiments, the incubation of cells with a selection reagent, such as an immunoaffinity-based selection reagent, is performed within the chamber.

[0330] In some embodiments, by performing such a selection process or part thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in a chamber cavity, the user can control certain parameters, such as the volume of various solutions, the addition of solutions during processing, and the timing thereof, which may offer advantages compared to other available methods. For example, by reducing the volume of liquid in the cavity during incubation, the concentration of particles used for selection (e.g., bead reagents) can be increased, thereby increasing the chemical potential of the solution without affecting the total number of cells in the cavity. This may, as a result, enhance the paired interaction between the cells being processed and the particles used for selection.

[0331] In some embodiments, performing the incubation process within a chamber allows, for example, if the chamber is associated with the systems, circuits, and control units described herein, the user can achieve agitation of the solution at a desired time during incubation, which can also improve the interaction.

[0332] In some embodiments, at least part of the selection process, including incubation with a cell selection reagent, is performed in a chamber. In some embodiments of such a process, a certain volume of cells is mixed with a much smaller amount of a selection reagent based on a desired affinity than would typically be used when performing a similar selection in a tube or container according to the manufacturer's instructions for selecting the same number and / or volume of cells. In some embodiments, an amount of selection reagent or a plurality of selection reagents is used that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the same amount of selection reagent used for cell selection in a tube or container-based incubation of the same number and / or volume of cells according to the manufacturer's instructions for use.

[0333] In some embodiments, for cell selection, e.g., selection based on immunoaffinity, cells are incubated in a composition within a chamber, which also contains a selection buffer along with a selection reagent, e.g., an antibody optionally bound to a surface marker on the cells to be concentrated and / or removed, but not to other surface markers on the cells in the composition, e.g., a scaffold, e.g., a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., monoclonal antibodies specific to CD4 and CD8. In some embodiments, as described, the selection reagent is added to the cells in the chamber cavity in substantially less than the amount of selection reagent typically used to achieve approximately the same or comparable selection efficiency for the same number or volume of cells when the selection is performed in a tube with shaking or rotation (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) compared to the amount of selection reagent required. In some embodiments, incubation is performed with the addition of selection buffer to cells and selection reagents to achieve a target volume in incubation of, for example, 10 mL to 200 mL, for example, at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagents, or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagents. In some embodiments, the selection buffer and selection reagents are pre-mixed before addition to cells. In some embodiments, the selection buffer and selection reagents are added to cells separately. In some embodiments, incubation for selection is performed periodically. The process is carried out under mild mixing conditions, which may help promote energetically favorable interactions, thereby achieving high selectivity and potentially allowing for the use of fewer selective reagents overall.

[0334] In some embodiments, the total incubation time with the selected reagent is approximately 5 minutes to 6 hours, for example, 30 minutes to 3 hours, for example, at least approximately 30 minutes, 60 minutes, 120 minutes, or 180 minutes, or at least approximately 30 minutes, 60 minutes, 120 minutes, or 180 minutes.

[0335] In some embodiments, incubation is typically carried out under mixed conditions, for example, with a relatively weak force or low speed, for example, a speed lower than the speed used to pelletize the cells, for example, 600 rpm to 1700 rpm or about 600 rpm to 1700 rpm (for example, 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). , or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, in the presence of centrifugation at RCF on the inner wall of a sample of 80 g to 100 g or about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g) or the inner wall of a chamber or other container. In some embodiments, centrifugation is performed using repeated pauses following such low-speed centrifugation time, e.g., centrifugation and / or pauses of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, e.g., centrifugation of about 1 or 2 seconds followed by pauses of about 5, 6, 7, or 8 seconds.

[0336] In some embodiments, such a process is carried out within a fully sealed system essential to the chamber. In some embodiments, this process (and in some embodiments, one or more additional steps, such as a washing step immediately before washing a sample containing cells, such as an apheresis sample) is carried out in an automated manner such that cells, reagents, and other components are injected into and extruded from the chamber at appropriate times, and centrifugation is achieved, so that the washing and binding steps are completed in a single sealed system using an automated program.

[0337] In some embodiments, after incubation and / or mixing of cells and a selection reagent and / or multiple selection reagents, the incubated cells are subjected to separation for selection of cells based on the presence or absence of a particular reagent or multiple reagents. In some embodiments, the separation is performed within the same closed system in which the incubation of cells and selection reagents was performed. In some embodiments, after incubation with the selection reagents, the incubated cells, which include cells bound to the selection reagents, are transferred to a system for separation of cells based on immunoaffinity. In some embodiments, the system for separation based on immunoaffinity is or includes a magnetic separation column.

[0338] In some embodiments, the isolation method includes the separation of different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids in cells. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some embodiments, the isolation includes the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds to such markers, usually followed by a washing step and separation of cells bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner.

[0339] Such separation steps may be based on positive selection, in which cells bound to the reagent are reserved for further use, and / or negative selection, in which cells that did not bind to the antibody or binding partner are reserved. In some examples, both fractions are reserved for further use.

[0340] In some embodiments, negative selection may be particularly useful, where antibodies that specifically identify cell types within a heterogeneous population are unavailable, so that separation is best performed based on markers expressed by cells other than the target population.

[0341] Separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, e.g., cells expressing a marker, means increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, means decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0342] In some cases, fractions positively or negatively selected in one step are subjected to multiple rounds of separation steps, such as subsequent positive or negative selection. In some cases, cells expressing multiple markers simultaneously can be removed in a single separation step by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed in various cell types.

[0343] For example, in some embodiments, specific subpopulations of T cells, e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. For example, CD3+, CD28+ T cells can be positively selected using anti-CD3 / anti-CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In some embodiments, the cell population is enriched with T cells having a naive phenotype (CD45RA+CCR7+).

[0344] In some embodiments, isolation is performed by enriching a specific cell population by positive selection or depleting a specific cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating cells with one or more antibodies or other conjugates that are expressed on the cells to be positively or negatively selected, or that specifically bind to one or more surface markers expressed at or at relatively higher levels (marker high) (marker+).

[0345] In certain embodiments, a biological sample, such as a PBMC or other leukocyte sample, is subjected to the selection of CD4+ T cells, ensuring both negative and positive fractions are obtained. In certain embodiments, CD8+ T cells are obtained from the negative fraction. In some embodiments, a biological sample is subjected to the selection of CD8+ T cells, ensuring both negative and positive fractions are obtained. In certain embodiments, CD4+ T cells are obtained from the negative fraction.

[0346] In some embodiments, T cells are isolated from a PBMC sample by negative selection of a marker expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. In some embodiments, a CD4+ or CD8+ selection step is used to isolate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations may be further sorted into subpopulations by positive or negative selection of a marker expressed on or relatively more highly expressed on one or more naive T cell, memory T cell, and / or effector T cell subpopulations.

[0347] In some embodiments, CD8+ cells are further enriched or removed, for example, by positive or negative selection based on surface antigens associated with each subpopulation, to include naive cells, central memory cells, effector memory cells, and / or central memory stem cells. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase efficacy, for example, to improve long-term survival, proliferation, and / or engraftment after administration, and in some embodiments, efficacy is particularly strong in such subpopulations. In some embodiments, efficacy is further enhanced by combining TcM-enriched CD8+ T cells and CD4+ T cells. In some embodiments, efficacy is enhanced by enriching T cells having a naive phenotype (CD45RA+CCR7+).

[0348] In this embodiment, memory T cells are present in both the CD62L+ subset and the CD62L subset of CD8+ peripheral blood lymphocytes. PBMCs can be enriched with CD62L CD8+ and / or CD62L+CD8+ fractions, or they can be removed, for example, using anti-CD8 and anti-CD62L antibodies.

[0349] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or surface high expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In some embodiments, this is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, isolation of the CD8+ population enriched with TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one embodiment, enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on the expression of CD14 and CD45RA, and positive selection based on CD62L. In some embodiments, such selections are performed simultaneously, and in other embodiments, they are performed sequentially in any order. In some embodiments, the same CD4 expression-based selection step used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, thereby ensuring that both positive and negative fractions from the CD4-based separation are available for use in subsequent steps of the method, optionally after one or more further positive or negative selection steps.

[0350] In certain cases, a PBMC sample or other leukocyte sample is subjected to selection of CD4+ cells, ensuring both negative and positive fractions are obtained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are performed in either order.

[0351] CD4+ T helper cells identify cell populations that possess cell surface antigens, The cells are sorted into naive cells, central memory cells, and effector cells. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L and CD45RO. In some embodiments, T cells with a naive phenotype are CD45RA+CCR7+.

[0352] In one example, a monoclonal antibody cocktail for enriching CD4+ cells by negative selection typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or solid matrix, such as magnetic or paramagnetic beads, to enable the separation of cells in positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques.

[0353] In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads or MACS beads). The magnetically responsive material, such as particles, is typically bound directly or indirectly to a binding partner, such as a molecule, or to an antibody that specifically binds to a surface marker present in cells, a group of cells, or a cell population that is to be separated, such as negative or positive selection.

[0354] In some embodiments, the magnetic particles or beads include a magnetically responsive material bound to a member that specifically binds, such as an antibody or other binding partner. Numerous well-known magnetically responsive materials exist that are used in magnetic separation methods.

[0355] Incubation is typically performed under conditions in which molecules such as antibodies or binding partners, or secondary antibodies or other reagents that specifically bind to such antibodies or binding partners, which are usually bound to magnetic particles or beads, specifically bind to cell surface molecules (if present on cells in the sample).

[0356] In some embodiments, a sample is placed in a magnetic field, and cells to which magnetically responsive or magnetizable particles are bound are attracted to the magnet and separated from unlabeled cells. Positive selection secures cells that are attracted to the magnet, while negative selection secures cells that are not attracted (unlabeled cells). In some embodiments, a combination of positive and negative selection is performed during the same selection process, where positive and negative fractions are secured and further processed or subjected to further separation steps.

[0357] In some embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are bound to cells by coating with a primary antibody specific to one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used together with a biotinylated primary or secondary antibody.

[0358] In some embodiments, the magnetically responsive particles remain bound to cells to be subsequently incubated, cultured, and / or manipulated, and in some embodiments, the particles remain bound to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive unlabeled antibodies and magnetizable particles or antibodies conjugated with a cleavable linker. In some embodiments, the magnetizable particles are biodegradable.

[0359] In some embodiments, affinity-based selection is performed by a magnetically activated cell sorter (MACS) (Miltenyi Biotech, Auburn, CA). The magnetically activated cell sorter (MACS) system enables high-purity selection of cells bound to magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells bound to magnetized particles are held in place, and unbound species are eluted. Then, after this initial elution step is complete, species that were trapped in the magnetic field and whose elution was prevented are released in several ways so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from heterogeneous cell populations.

[0360] In some embodiments, isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the Method. In some embodiments, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination. In one example, the system is such as that described in International Publication No. 2009 / 072003 or U.S. Patent Application Publication No. 20110003380(A1).

[0361] In some embodiments, the system or apparatus performs one or more, for example, all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and / or computer program communicating with the system or apparatus, which enables the user to program, control, evaluate the results of, and / or adjust various embodiments of the processing, isolation, manipulation, and formulation steps.

[0362] In some embodiments, separation and / or other steps are performed using the CliniMACS system (Miltenyi Biotec) for automated cell separation at a clinical scale within a closed, sterile system. Components may include an embedded microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the embedded microcomputer controls all components of the instrument and instructs the system to perform repetitive procedures in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for the selective column. The peristaltic pump controls the flow rate within the tubing set and, together with the pinch valves, ensures a controlled flow of buffer and continuous suspension of cells through the system.

[0363] In some embodiments, the CliniMACS system uses magnetizable particles coupled to antibodies, supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tube set, which is then connected to a buffer bag and a cell collection bag. The tube set consists of pre-assembled sterile tubes containing a pre-column and a separation column, and is intended for single use only. After the program starts, the system automatically applies the cell sample onto the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population used in the method described herein is unlabeled and is not retained in the column. In some embodiments, the cell population used in the method described herein is labeled and is retained in the column. In some embodiments, the cell population used in the method described herein is eluted from the column after the magnetic field is removed and collected in a cell collection bag.

[0364] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system includes a cell processing unit that enables automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of cell products from the source. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The Prodigy system may also include an integrated cell culture chamber for performing cell culture protocols such as cell differentiation and proliferation, antigen loading, and long-term cell culture. An inlet may allow for sterile removal and replenishment of the culture medium, and cells may be monitored using an integrated microscope.

[0365] In some embodiments, the cell populations described herein are collected and concentrated (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid flow. In some embodiments, the cell populations described herein are collected and concentrated (or depleted) by sorting on a preparative scale (FACS). In certain embodiments, the cell populations described herein are collected and concentrated (or depleted) by the use of a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, for example, International Publication No. 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376). In any case, the cells may be labeled with multiple markers, which allows for the isolation of a clearly defined T cell subset with high purity.

[0366] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation in positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on the binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid flow by, for example, fluorescence-activated cell sorting (FACS) and / or a microelectromechanical system (MEMS) chip combined with a flow cytometry detection system, for example. Such methods enable simultaneous positive and negative selection based on multiple markers.

[0367] In some embodiments, the preparation method includes a step of freezing the cells, e.g., for cryopreservation, either before or after isolation, incubation, and / or processing. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and some monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution after a washing step to remove, for example, plasma and platelets. In some embodiments, any of various known freezing solutions and parameters may be used. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 in the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen at -80°C at a rate of 1°F. It is frozen to °C and stored in the gas phase of a liquid nitrogen storage tank.

[0368] In some embodiments, isolation and / or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more distinct input compositions are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, distinct input compositions are isolated, selected, enriched, and / or obtained from distinct biological samples collected, harvested, and / or obtained from the same subject.

[0369] In certain embodiments, one or more input compositions are or comprise of enriched T cell compositions containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD3+ T cells. In one embodiment, the enriched T cell input composition is essentially composed of CD3+ T cells.

[0370] In certain embodiments, one or more input compositions are or include enriched CD4+ T cell compositions containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In certain embodiments, the CD4+ T cell input composition contains and / or does not contain CD8+ T cells, and / or does not contain or substantially contains CD8+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD4+ T cells.

[0371] In certain embodiments, one or more compositions are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells, or compositions of CD8+ T cells containing or comprising such. In certain embodiments, the CD8+ T cell composition contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells and / or does not contain CD4+ T cells and / or does not contain or substantially contains CD4+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD8+ T cells.

[0372] In some embodiments, cells are incubated and / or cultured before or in conjunction with genetic manipulation. The incubation step may include culture, cultivation, stimulation, activation, and / or proliferation. Incubation and / or operations may be performed in a culture vessel, e.g., a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culture or culturing cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include conditions designed to induce proliferation, growth, activation, and / or survival of cells in a population, conditions designed to mimic antigen exposure, and / or conditions designed to prepare cells for genetic manipulation, e.g., introduction of recombinant antigen receptors. Conditions include specific media, temperature, oxygen content, carbon dioxide content, time, drugs, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulants, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors. It may contain one or more other drugs designed to activate the body and cells.

[0373] In some embodiments, the stimulating condition or stimulant includes one or more agents, for example, ligands that can stimulate or activate the intracellular signaling domain of the TCR complex. In some embodiments, the agent activates or induces the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies specific to the TCR, such as anti-CD3 antibodies. In some embodiments, the stimulating condition includes one or more agents, for example, ligands that can stimulate costimulatory receptors, such as anti-CD28. In some embodiments, such agents and / or ligands may be bound to a solid support such as beads, and / or one or more cytokines. Optionally, the growth method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (for example, at a concentration of at least about 0.5 ng / mL). In some embodiments, the stimulant includes IL-2, IL-15, and / or IL-7. In some embodiments, the IL-2 concentration is at least about 10 units / mL. In some embodiments, incubation is carried out according to the techniques described in U.S. Patent No. 6,040,177 by Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0374] In some embodiments, T cells are grown by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to a culture initiation composition (for example, the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population being grown), and incubating the culture (for example, for a time sufficient to increase the number of T cells). In some embodiments, non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000–3600 rad to inhibit cell division. In some embodiments, the feeder cells are added to the culture medium before the addition of the T cell population.

[0375] In some embodiments, the stimulation conditions include a temperature suitable for the proliferation of human T lymphocytes, e.g., at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, generally 37 degrees Celsius, or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-mitotic EBV-transformed lymphoblast-like cells (LCLs) as feeder cells. The LCLs may be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some embodiments, the LCL feeder cells are provided in any preferred amount, e.g., at least about 10:1 LCL feeder cells to initial T lymphocytes.

[0376] In the embodiment, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with the antigen. For example, an antigen-specific T cell line or clone against a cytomegalovirus antigen can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0377] In some embodiments, at least part of the incubation under one or more stimulating conditions or in the presence of stimulating agents is performed, for example, in the internal cavity of a centrifugal chamber under centrifugal rotation, as described, for example, in International Publication WO2016 / 073602. In some embodiments, at least part of the incubation performed in the centrifugal chamber includes mixing with a reagent or a number of reagents to induce stimulation and / or activation. Several embodiments In this configuration, cells, such as selected cells, are subjected to stimulating conditions or mixed with stimulating agents in a centrifugation chamber. In some embodiments of this process, a certain volume of cells is mixed with one or more stimulating conditions or agents in much smaller quantities than would typically be used when performing similar stimulation on a cell culture plate or other system.

[0378] In some embodiments, the stimulant is added to the cells in the chamber cavity in a substantially smaller amount (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) compared to the amount of stimulant typically used to achieve approximately the same or similar selection efficiency for the same number or volume of cells, when selection is performed without mixing and with periodic shaking or rotation in the chamber, e.g., tube or bag. In some embodiments, incubation is carried out with the addition of incubation buffer to cells and stimulants to achieve a target volume in incubation of, for example, 10 mL to 200 mL, for example, at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagents, or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagents. In some embodiments, the incubation buffer and stimulants are pre-mixed before being added to the cells. In some embodiments, the incubation buffer and stimulants are added to the cells separately. In some embodiments, incubation for stimulation is carried out under periodic, gentle mixing conditions, which may help promote energetically favorable interactions, thereby achieving cell stimulation and activation while allowing for the use of less stimulant overall.

[0379] In some embodiments, incubation is typically carried out under mixed conditions, for example, with a relatively weak force or low speed, for example, a speed lower than the speed used to pelletize the cells, for example, 600 rpm to 1700 rpm or about 600 rpm to 1700 rpm (for example, 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). , or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, in the presence of centrifugation at RCF on the inner wall of a sample of 80 g to 100 g or about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g) or the inner wall of a chamber or other container. In some embodiments, centrifugation is performed using repeated pauses following such low-speed centrifugation time, e.g., centrifugation and / or pauses of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, e.g., centrifugation of about 1 or 2 seconds followed by pauses of about 5, 6, 7, or 8 seconds.

[0380] In some embodiments, for example, the total duration of incubation with the stimulant is 1 to 96 hours, 1 to 72 hours, 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours, or approximately 1 to approximately 96 hours, approximately 1 to approximately 72 hours, approximately 1 to approximately 48 hours, approximately 4 to approximately 36 hours, approximately 8 to approximately 30 hours, or approximately 12 to approximately 24 hours, for example, at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours, or at least approximately 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, further incubation is 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours, or approximately 1 to approximately 48 hours, approximately 4 to approximately 36 hours, approximately 8 to approximately 30 hours, or approximately 12 to approximately 24 hours (including bot...

Claims

1. A method for determining the risk of adverse events during the administration of immunotherapy to cancer patients requiring immunotherapy, wherein the method is: Quantifying baseline serum levels of one or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET from the cancer subjects, or To quantify the day 0 serum levels of one or more biomarkers selected from SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5 from the cancer subjects, The process includes determining the risk of the adverse event in the cancer subject based on the step of quantifying the baseline serum level of one or more biomarkers, or the step of quantifying the day 0 serum level of one or more biomarkers from the cancer subject, At least one of the following is correlated with an increased risk of the adverse event in the cancer patient: an increase in the baseline serum level of one or more biomarkers relative to the control baseline serum level of one or more biomarkers and / or an increase in the day 0 serum level of one or more biomarkers relative to the control day 0 serum level of one or more biomarkers. The method wherein the adverse event is one or more of the following: immune activation-induced stress, grade 3 + CRS, and grade 3 + NE.

2. The aforementioned method, To quantify the baseline serum levels of one or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET from the cancer subjects, To quantify the day 0 serum levels of one or more biomarkers selected from SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5 from the cancer subjects, The method according to claim 1, comprising the steps of quantifying the baseline serum level of one or more biomarkers, and determining the risk of the adverse event in the cancer subject based on the steps of quantifying the day 0 serum level of one or more biomarkers from the cancer subject.

3. The method according to claim 1 or 2, comprising quantifying baseline serum levels of two or more biomarkers selected from REG3A, KYNU, OSMR, NELL2, and MET from the cancer subject.

4. The method according to any one of claims 1 to 3, comprising quantifying the day 0 serum levels of two or more biomarkers selected from SOD2, VAMP5, PCDH17, ACE2, REG1B, REG3A, AREG, CELA3A, CEACAM1, STK11, LY9, LY96, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCDH17, TGFB1, ADAM15, BSG, HLA-DRA, ICAM2, OSMR, SERPINB9, CCL16, MEGF9, and MFAP5 from the cancer subject.

5. The method according to any one of claims 1 to 4, wherein the control baseline serum level of the one or more biomarkers is a baseline serum level observed as a history of the one or more biomarkers that has not been previously observed to be associated with the development of the adverse event, and the control day 0 serum level of the one or more biomarkers is a day 0 serum level observed as a history of the one or more biomarkers that has not been previously observed to be associated with the development of the adverse event.

6. If the baseline serum level of one or more biomarkers selected from MET, OSMR, NELL2, REG3A, and KYNU, and the day 0 serum level of one or more biomarkers selected from AREG, CKAP4, CXCL1, EIF5A, IL1A, KIFBP, KIRREL2, NUB1, PAG1, PCHD17, STK11, TGFB1, ACE2, ADAM15, BSG, CEACAM1, HLA-DRA, ICAM2, LY9, LY96, OSMR, REG3A, SERPINB9, SOD2, VAMP5, CCL16, CELA3A, MEGF9, MFAP3, and REG1B exceed a predetermined normalized protein expression range for one or more of the aforementioned cancer subjects, then the risk of the aforementioned adverse events is determined to be increased. The predetermined normalized protein expression ranges for one or more of the biomarkers are: MET: 0.56-0.75, OSMR (baseline): 0.62-0.77, NELL2: 0.14-0.50, REG3A (baseline): 1.80-2.19, KYNU: 1.60-1.98, CCL16: 0.84-1.25, CELA3A: 1.37-2.01, ME GF9: 0.26–0.48, MFAP3: 0.33–0.49, REG1B: 1.49–1.99, AREG: 0.88–1.22, BSG: 0.73–0.96, CKAP4: 1.83–2.31, CXCL1: 3.48–3.91, EIF5A: 0.09–0.23, IL1A: 0.46–0.81, KIFBP: 1.36–1.66, KIR For REL2: 1.62–2.13, for NUB1: 1.39–1.77, for OSMR (Day 0): 0.65–0.79, for PAG1: 2.16–2.74, for PCDH17: 1.03–1.32, for STK11: 1.09–1.35, for ACE2: 1.20–1.65, for ADAM15: 1.09–1.31, for CEACAM1: 0.68–0.90, for HLA-DRA: The method according to any one of claims 1 to 5, wherein the values ​​are 1.13 to 1.41 for ICAM2, 1.21 to 1.43 for LY9, 0.11 to 0.49 for REG3A (day 0), 2.34 to 2.88 for SERPINB9, 2.12 to 2.30 for SOD2, 1.37 to 1.86 for LY96, 0.54 to 0.70 for TGFB1, and 1.30 to 1.73 for VAMP5.

7. The method according to any one of claims 1 to 6, further comprising, if the cancer subject is determined to be at increased risk of adverse events, treating the cancer subject with a preconditioning regimen modified from a standard preconditioning regimen; not administering the immunotherapy to the cancer subject; administering the immunotherapy to the cancer subject at a dose modified from a standard dose; or administering the immunotherapy to the cancer subject in combination with an agent for mediating immune activation-inducing stress, grade 3 + CRS, and / or grade 3 + NE.

8. The method according to claim 7, wherein the modified preconditioning regimen comprises administering to the cancer subject at least one modified dose of cyclophosphamide and fludarabine instead of a predetermined dose of cyclophosphamide or fludarabine administered to a control subject.

9. If the aforementioned cancer patient is determined to be at increased risk of adverse events, the immunotherapy may be administered. The method according to any one of claims 1 to 8, further comprising administering it in combination with a combination therapy comprising an agent that reduces cytokine induction and / or endothelial cell destruction.

10. The method according to claim 9, wherein the drug reduces cytokine induction.

11. The method according to claim 10, wherein the drug is administered to the cancer target before the administration of the immunotherapy, before the peak expansion of the immunotherapy, or at the time of the peak expansion of the immunotherapy.

12. The method according to any one of claims 9 to 11, wherein the agent is selected from an anti-IL-1 molecule, a T cell activation inhibitor, a JAK inhibitor, an anti-GM-CSF molecule, an anti-TNF molecule, an Ang2 inhibitor, an anti-angiogenic therapy, and an anti-IFNg molecule.

13. The method according to any one of claims 1 to 12, wherein the immunotherapy is CAR T cell therapy, TCR T cell therapy, tumor-infiltrating lymphocyte (TIL) cell therapy, or bispecific T cell engager (BiTE) therapy.

14. The method according to claim 13, wherein the immunotherapy is autologous or allogeneic.

15. The method according to claim 13, wherein the immunotherapy is CAR T or TCR T cell therapy that recognizes a target antigen.

16. 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 (IGFl)-l, 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 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 antigen, e.g., HIV-specific antigen (e.g., H; The method according to claim 15, wherein the tumor antigen is selected from IV gp120, GPC3 (glypican 3), and any derivative or variant of these antigens.

17. The aforementioned cancers include 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, One or more of the following: 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, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance (MGUS), plasmacytoplasmic 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, asymptomatic myeloma (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), monoclonal hypergammaglobulinemia of unknown significance (MGUS), plasmacytoplasmic leukemia The method according to any one of claims 1 to 16, wherein the cancer or tumor is selected from ma 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.

18. The method according to claim 17, 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, DLBCL arising from follicular lymphoma, or mantle cell lymphoma.

19. The immunotherapy according to any one of claims 1 to 18, wherein the immunotherapy is selected from axicapbutagen silolucel, brexcapbutagen autolucel, tisagenlecleucel, lysocabbutagen maralucel, and bb2121.

20. A method for determining the risk of adverse events during the administration of immunotherapy to cancer patients requiring immunotherapy, wherein the method is: Determining baseline serum levels and / or day 0 serum levels of multiple biomarkers from the aforementioned cancer subjects, At least in part, the weight for each of the multiple biomarkers is determined by inputting the baseline serum levels of the multiple biomarkers into the algorithm, This includes determining the probability of the risk value of the adverse event based at least in part on the weights for each of the plurality of biomarkers, If the probability of the aforementioned risk value is at least the same as a predetermined cutoff value, then the subject has an increased risk of the adverse event compared to the control group. The method comprising the aforementioned multiple biomarkers, ACE2, IL1A, SERPINB9, and LY96.

21. The method according to claim 20, wherein the predetermined cutoff is a Youden cutoff value of 0.10 to 0.

35.

22. The method according to claim 20, wherein the algorithm includes a logical regression model, a random forest algorithm, or a regularized gradient boosting framework algorithm.

Images