Chimeric receptor t cell therapy using properties of the tumor microenvironment

Abstract

To provide a method for treating malignant tumors.SOLUTION: A method of treating a malignancy in a patient, the method comprising: (a) analyzing a tumor biopsy from the patient to characterize a tumor microenvironment; and (b) administering to the patient an effective dose of a T cell comprising one or more chimeric receptors, wherein the effective dose is determined using the characteristics of the tumor microenvironment.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 656,825, filed April 12, 2018, U.S. Provisional Patent Application No. 62 / 827,770, filed April 1, 2019, and U.S. Provisional Patent Application No. 62 / 831,946, filed April 10, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 11, 2019, is named K-1065_01_SL.txt and is 8 kilobytes in size. [Background technology]

[0003] Human cancers are originally composed of normal cells that undergo genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that are distinctly 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 utilize various mechanisms to prevent immune cells, such as T lymphocytes and B lymphocytes, from successfully targeting the cancer cells.

[0004] Human T cell therapy relies on enriched or engineered human T cells to target and kill cancer cells in patients. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct T cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which contain binding domains that can interact with specific tumor antigens, enable T cells to target and kill cancer cells that express specific tumor antigens. Summary of the Invention

[0005] Tumor microenvironment (TME) characteristics prior to CAR T cell infusion can influence clinical outcomes. The present disclosure provides methods to assess the TME and correlate it with outcome using immune-related gene expression signatures of the TME and / or intratumoral T cell density.

[0006] Each of the aspects and embodiments described below can be combined unless the context clearly dictates otherwise.

[0007] In one aspect, the present disclosure provides a method of treating a malignant tumor in a patient, the method comprising: (a) analyzing a tumor biopsy from the patient to characterize the tumor microenvironment; and (b) administering to the patient an effective dose of T cells comprising one or more chimeric receptors, wherein the effective dose is determined using the characteristics of the tumor microenvironment.

[0008] In some embodiments, the tumor microenvironment is characterized using gene expression profiling. In some embodiments, the tumor microenvironment is characterized based on intratumoral T cell density. In some embodiments, the tumor microenvironment is characterized using gene expression profiling and intratumoral T cell density.

[0009] In some embodiments, gene expression profiling involves the use of a specific gene panel. In some embodiments, the panel includes measuring the expression levels of CD3G, STAT4, CD3E, CD3D, GZMK, GZMM, PRF1, CD8A, ICOS, CXCL10, STAT1, IL15, CCR2, CCL2, IRF1, TBX21, GZMA, CXCR3, GZMB, CD69, CXCL11, and combinations thereof. In some embodiments, the panel includes CTLA4, GZMH, CD8A, PDCD1, CD3G, IRF1, CX3CL1, TNFRSF9, CD3E, GZMA, CXCL10, TSLP, REN, GZMB, TNFRSF18, CCL2, GZMK, CXCL11, CD69, CD247, CCL5, STAT4, CD274, GNLY, ITGAE, LAG3, IL15, LTK, PRF1, CD3D, PF4, TBX21, ICOS, CXCL9, IFNG, VEGFA, STAT1, GZMM, CXCL13, CXCR3, CCR2, IL17A, PROM1, and combinations thereof. Includes Profiling Panel.

[0010] In some embodiments, the method comprises measuring the expression level of one or more genes selected from CD3G, STAT4, CD3E, CD3D, GZMK, GZMM, PRF1, CD8A, ICOS, CXCL10, STAT1, IL15, CCR2, CCL2, IRF1, TBX21, GZMA, CXCR3, GZMB, CD69, CXCL11, and combinations thereof.

[0011] In some embodiments, the method comprises measuring the expression level of one or more genes selected from CTLA4, GZMH, CD8A, PDCD1, CD3G, IRF1, CX3CL1, TNFRSF9, CD3E, GZMA, CXCL10, TSLP, REN, GZMB, TNFRSF18, CCL2, GZMK, CXCL11, CD69, CD247, CCL5, STAT4, CD274, GNLY, ITGAE, LAG3, IL15, LTK, PRF1, CD3D, PF4, TBX21, ICOS, CXCL9, IFNG, VEGFA, STAT1, GZMM, CXCL13, CXCR3, CCR2, IL17A, PROM1, and combinations thereof.

[0012] In some embodiments, the method comprises measuring the expression level of a gene selected from a PanCancer Immune Profiling Panel.

[0013] In some embodiments, the method comprises measuring the expression level of a B cell marker, hi some embodiments, the B cell marker is selected from BLK, CD19, CR2, MS4A1, TNFRSF17, and combinations thereof.

[0014] In some embodiments, the method comprises measuring the expression level of a T cell marker, hi some embodiments, the T cell marker is selected from CD2, CD2E, CD3G, CD6, and combinations thereof.

[0015] In some embodiments, the method comprises measuring the expression level of a specific gene panel comprising genes associated with the innate immune response, hi some embodiments, the specific gene panel comprises markers of cytotoxic cells, dendritic cells, macrophages, granulocytes, and combinations thereof.

[0016] In some embodiments, the particular gene panel comprises genes selected from CD8, BLC2, and combinations thereof.

[0017] In some embodiments, the specific gene panel includes CCL12, CCL17, and It includes genes selected from these combinations.

[0018] In some embodiments, the particular gene panel includes genes selected from APOE, CCL7, and combinations thereof.

[0019] In some embodiments, the particular gene panel comprises genes selected from CMA1, CSF3R, and combinations thereof.

[0020] In some embodiments, the method comprises measuring the expression level of one or more genes selected from CTLA4, CD3g, CD3e, CD27, SH2B2, ICOSL, and combinations thereof.

[0021] In some embodiments, the method comprises measuring the expression level of one or more genes selected from CD27, SH2B2, ICOSLG, HLA-DQA1, HLA-DQB1, MAGEB2, PRAME, MAGEA1, IL22RA1, SSX1, CCL20, NEFL, C9, GZMM, KIR Act subgroup 2, HLA-DOB, and combinations thereof.

[0022] In some embodiments, the method comprises measuring the expression level of one or more genes selected from CD27, SH2B2, ICOSLG, and combinations thereof.

[0023] In some embodiments, the method comprises measuring the expression level of one or more genes selected from HLA-DQA1, HLA-DQB1, MAGEB2, PRAME, MAGEA1, IL22RA1, SSX1, CCL20, NEFL, C9, GZMM, KIR Act subgroup 2, HLA-DOB, and combinations thereof.

[0024] In some embodiments, the method includes measuring the density of T cells in the tissue microenvironment (i.e., intratumoral T cell density), for example, by immunohistochemical staining of a tumor biopsy. In some embodiments, the method includes measuring the density of CD3 T cells in the tissue microenvironment (i.e., intratumoral T cell density), for example, by immunohistochemical staining of a tumor biopsy. + T cells and / or CD8 + This involves measuring the density of T cells.

[0025] In some embodiments, the method includes determining an immune score based on the gene expression profile. In some embodiments, the method includes determining an immune score based on intratumoral T cell density. In some embodiments, the method includes adjusting the total dose of CAR-T cells using the immune score. In some embodiments, the method includes providing an immunomodulatory compound or immunomodulatory intervention to increase the immune score before administering the CAR-T cells.

[0026] In some embodiments, the effective dose is at least 1 x 10 per kg of body weight. 6 Contains CAR-positive viable T cells.

[0027] In some embodiments, the chimeric receptor targets a tumor antigen, such as tumor-associated surface antigens, e.g., 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, pancreatic ductal epithelial mucin, EBV-specific antigen, or the like. Heteroantigens, 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, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen , human telomerase reverse transcriptase, Insulin Growth Factor IgF1 receptor, IGF-II, IL-11R alpha, IL-13R-a2, influenza virus specific antigen, CD38, insulin growth factor (IGF1)-1, intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa fever virus specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutated hsp70-2, mutant p53, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate-specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate-specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, surviving and telomerase, TAG-72, extra domain A of fibronectin The target is a tumor antigen selected from the group consisting of extradomain A (EDA) and extradomain B (EDB), as well as the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gp120), and any derivatives or mutants of these surface markers.

[0028] In some embodiments, the chimeric receptor specifically targets CD19.

[0029] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the chimeric receptor is a T cell receptor (TCR).

[0030] In some embodiments, the malignant tumor is selected from the group consisting of solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large cell lymphoma (DLC), and leukemia. Diffuse large B cell lymphoma (DLBCL), follicular lymphoma ( FL), transformed follicular lymphoma, splenic marginal zone lymphoma 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, one or more of B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, manleukemia, Toll cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., dysplasmocytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, and P (also known as EP syndrome), or a combination thereof.

[0031] In some embodiments, the malignant tumor is diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, non-Hodgkin's lymphoma, metastatic melanoma, transformed follicular lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma.

[0032] In some embodiments, the effective dose is optimized to increase the likelihood that the patient will respond to treatment.

[0033] In some embodiments, a tumor biopsy is obtained from the patient prior to treatment with chimeric receptor therapy.

[0034] In one aspect, the present disclosure includes a method of determining whether a patient will respond to chimeric receptor therapy, the method comprising: (a) analyzing a tumor biopsy from the patient to characterize the tumor microenvironment using a gene expression profile; (b) determining an immune score based on the gene expression profile; and (c) determining whether the patient will respond to chimeric receptor therapy based on the immune score.

[0035] In one aspect, the disclosure provides a method for determining whether a patient will respond to chimeric receptor therapy, comprising: (a) analyzing a tumor biopsy from the patient to determine the level of T cells (e.g., CD3 + T cells and / or CD8 + (b) characterizing the tumor microenvironment by quantifying intratumoral T cell density; (c) determining an immune score based on the intratumoral T cell density; and (d) determining whether the patient will respond to chimeric receptor therapy based on the immune score.

[0036] In one aspect, the present disclosure provides a method of treating a malignant tumor in a patient, the method comprising: (a) analyzing a tumor biopsy from the patient prior to chimeric receptor therapy to characterize the tumor microenvironment; (b) determining whether the patient will respond to chimeric receptor therapy based on the characteristics of the tumor microenvironment; and (c) administering to the patient an effective dose of T cells comprising one or more chimeric receptors, wherein the effective dose is determined using the characteristics of the tumor microenvironment.

[0037] In some embodiments, the method further comprises using characteristics of the tumor microenvironment to determine whether an additional therapeutic agent improves clinical efficacy. In some embodiments, the chimeric receptor therapy is administered with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered in combination with the chimeric receptor therapy. In some embodiments, the additional therapeutic agent is administered before or after the chimeric receptor therapy.

[0038] In some embodiments, the method further comprises administering a cytokine therapy, hi some embodiments, the cytokine therapy is IL-2 or IL-15.

[0039] In some embodiments, the method further comprises administering a stimulatory antibody, hi some embodiments, the stimulatory antibody is anti-41BB or anti-OX-40.

[0040] In some embodiments, the method further comprises administering a checkpoint blockade therapy, hi some embodiments, the checkpoint blockade therapy comprises CTLA4 or PD-1.

[0041] In some embodiments, the method further comprises administering an innate immune stimulator, hi some embodiments, the innate immune stimulator comprises a TLR agonist or a STING agonist.

[0042] In some embodiments, gene expression profiling involves measuring expression levels of proliferation markers, inflammatory markers, immunoregulatory markers, effector and / or chemokines. In some embodiments, the method involves measuring expression levels of one or more genes selected from IL-6, CRP, SAA, IL-5, ferritin, IL-1Ra, IL-2Rα, and combinations thereof. In some embodiments, the method involves measuring expression levels of one or more genes selected from GM-CSF, IFN-γ, IL-10, and combinations thereof. In some embodiments, the method involves measuring expression levels of one or more genes selected from IL-8, IP-10, MCP-1, and combinations thereof. In some embodiments, the method involves measuring expression levels of granzyme B. In some embodiments, the method involves measuring expression levels of CD3ε, CD28, and CTLA4. In some embodiments, the method involves measuring expression levels of MX1, ISG15, and MYD88. In some embodiments, the method comprises measuring expression levels of CD19, CD79B, and PAX5. In some embodiments, the method comprises measuring expression levels of PD-L1 and / or CD19. In some embodiments, the tumor biopsy is obtained prior to treatment with CAR T-cell therapy. In some embodiments, the tumor biopsy is obtained after treatment with CAR T-cell therapy. In some embodiments, the method comprises obtaining a tumor biopsy prior to treatment with CAR T-cell therapy and obtaining a tumor biopsy after treatment with CAR T-cell therapy. In some embodiments, the tumor biopsy is obtained 7 days, 14 days, 21 days, or 28 days after treatment with CAR T-cell therapy.

[0043] The drawings are for illustrative purposes only and are not limiting. [Brief explanation of the drawings]

[0044] [Figure 1]FIG. 1 shows a schematic diagram of the chimeric antigen receptor (CAR) configuration. [Figure 2] Figure 1 shows a schematic diagram of the ZUMA-1 clinical trial of patients receiving axi-cabutagen-ciloleucel. AE is adverse event, axi-cel is axi-cabutagen-ciloleucel, CAR is chimeric antigen receptor, CR is complete response, CRS is cytokine release syndrome, DLBCL is diffuse large B-cell lymphoma, NE is neurological event, ORR is objective response rate, PMBCL is primary mediastinal B-cell lymphoma, and TFL is transformed follicular lymphoma. [Figure 3] FIG. 1 shows a schematic diagram of the ZUMA-1 clinical trial protocol and timing of paired biopsies. [Figure 4] FIG. 1 shows the Immunosign™ clinical research assay panel used to assess key immune pathways within the tumor microenvironment. [Figure 5] Figure 5A shows the association between Immunosign™ 21 scores and clinical outcomes, as measured on samples from 25 patients treated with axicabtagene-ciloleucel (axi-cel) at a minimum follow-up of 9 months. One patient subsequently transitioned from a "non-responder" to a "responder" at 12 months of follow-up. Figure 5B shows the proportion of patients with high and low baseline Immunosign™ 21 scores who responded to axi-cel treatment. Figure 5C shows the proportion of patients with high and low baseline Immunosign™ 21 scores who did not respond to axi-cel treatment. The cutoff for high / low Immunosign™ 21 scores was defined as the 25th percentile of scores observed across samples. The stippled images in Figures 5B and 5C indicate low Immunosign™ 21 scores. [Figure 6]Figure 1 shows the difference in expression of the Immunosign™ 21 gene in responders compared to non-responders performed on samples from 25 patients treated with axicabtagene ciloleucel (axi-cel) with a minimum follow-up period of 9 months. [Figure 7] Figures 7A-7C show the association of the top three immune environment genes, CTLA4 (Figure 7A), CD3γ (Figure 7B), and CD3ε (Figure 7C), elevated in tumors from responders from a predefined panel of 43 immune genes performed on samples from 25 patients treated with axi-cel with a minimum follow-up period of 9 months. [Figure 8] Figure 8A shows the association between mean percentile Immunoscore™ performed on samples from treated patients and clinical outcome. Figure 8B shows the proportion of clinical outcomes observed among patients with a high baseline Immunoscore™. Figure 8C shows the proportion of clinical outcomes observed among patients with a low baseline Immunoscore™. The cutoff for high / low Immunosign™ 21 scores was defined as the median score observed among samples. CR is complete response, PR is partial response, SD is stable disease, and PD is progressive disease. [Figure 9] 9A and 9B show the densities of CD3+ and CD8+ cells, respectively, among complete responders and patients who showed other than complete responses in baseline biopsy sections from treated patients. [Figure 10] FIG. 1 shows a plot of the correlation between Immunosign™ 21 scores and Immunoscore™ assessed from baseline biopsies from treated patients. [Figure 11]Figure 1 shows pretreatment gene expression analysis in the TME of T cell-related genes (CD3ε, CD28, and CTLA4), innate immunity-related genes (MX1, ISG15, and MYD88), and B cell-related genes (CD19, CD79B, and PAX5) and clinical outcomes during a 1-year follow-up period of axi-cel treatment. CR is complete response, PR is partial response, SD is stable disease, and PD is progressive disease. [Figure 12] Figure 1 shows fold change in TME gene expression after 7-21 days of CAR-T treatment showing relative changes compared to baseline in immune inhibitory checkpoints (PD-L1, CTLA4, LAG3, TNFRSF18, ICOS), IFN-related genes (IRF1, STAT1, STAT4, IFNγ) and chemokines (CXCL9, CCL2, CCL5), effectors (CD8A, GNLY, GZMA, GZMM, GZMB, GZMH) and proliferation marker IL-15. [Figure 13] Figures 13A and 13B show nanostring analysis of differential changes in IL-15 (Figure 13A) and PD-L1 (Figure 13B). Pre-Tx is before treatment, W1-2 is weeks 1-2, and W4 is week 4. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present disclosure relates to methods of treating malignant tumors in patients using characteristics of the tumor microenvironment of a patient biopsy. The present disclosure is based, in part, on the surprising discovery that characteristics of the tumor microenvironment of a patient biopsy obtained prior to chimeric receptor therapy can be used to predict clinical outcome. As described herein, the tumor microenvironment profile prior to chimeric receptor therapy is used to determine an effective dose that impacts clinical outcome of chimeric receptor (e.g., CAR or TCR) T-cell therapy.

[0046] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0048] Unless specifically stated or clear from context, the term "or" as used herein is understood to include and encompass both "or" and "and."

[0049] As used herein, the term "and / or" is understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0050] The terms "for example" and "i.e." as used herein are used by way of example only, without any limitation, and should not be construed as referring only to those items expressly listed herein.

[0051] Terms such as "more than," "at least," and "more than," e.g., "at least one," are used to mean, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, This is understood to include 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the values ​​indicated, and also includes any higher number or fractional number in between.

[0052] Conversely, the term "less than" includes every value less than the indicated value. For example, "100 nucleotides or less" 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, 67, 68, 69 ... 4 pieces, 63 pieces, 62 pieces, 61 pieces, 60 pieces, 59 pieces, 58 pieces, 57 pieces, 56 pieces, 55 pieces, 54 pieces, 53 pieces, 52 pieces, 51 pieces, 50 pieces, 49 pieces, 48 ​​pieces, 47 pieces, 46 pieces, 45 pieces, 44 pieces, 43 pieces, 42 pieces, 41 pieces, 40 pieces, 39 pieces, 38 pieces, 37 pieces, 36 pieces, 35 pieces, 34 pieces, 33 pieces, 32 pieces, 31 pieces, 30 pieces, 29 pieces, 28 pieces, 27 pieces, 26 pieces, 25 pieces, 24 pieces, 23 pieces , 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, as well as any smaller number or fraction in between.

[0053] Terms such as "plurality," "at least two," "two or more," "at least a second," and the like, include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, This is understood to include 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 5000 or more, and also includes any higher number or fraction therebetween.

[0054] Throughout this specification, the words "comprising" or variations such as "comprises" or "comprising" mean that the elements indicated, integers or integers, are all elements that are part of the invention. It is understood that any reference herein implies the inclusion of an element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps. Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0055] Unless specifically stated or clear from context, the term "about" as used herein refers to a value or composition that falls within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. "About" or "approximately" can mean within a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to include a range that is 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 indicated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times a value. When a specific value or composition is provided in this disclosure, unless otherwise indicated, the meaning of "about" or "approximately" is intended to include an acceptable range of error for that specific value or composition.

[0056] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0057] Units, prefixes, and symbols used herein are presented in their accepted form in the Systeme International de Unites (SI). Numerical ranges are expressed in the units defining the range. Contains the number that specifies the

[0058] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. See, for example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press, "The Dictionary of Cell & Molecular Biology", 5th ed., (2013), Academic Press, and "The Oxford Dictionary of Biochemistry and Molecular Biology", Cammack et al. eds., 2nd ed, (2006), Oxford University Press, provide those of skill in the art with a general dictionary of many of the terms used in this disclosure.

[0059] "Administering" can be carried out in a variety of ways known to those skilled in the art. "Parenteral administration" refers to the physical introduction of an agent into a subject using any of a variety of delivery systems. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulations are administered by a route other than parenteral, such as orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical, and administration may be, for example, single, multiple, and / or over one or more extended periods of time.

[0060] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, antibodies may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, inter-connected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, namely, 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, namely, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). VH and VL each contain three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0061] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (referred to herein as "antibody Antibodies include antibodies, such as Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), mitochondria, and the like. These may include minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, antibodies as described herein refer to polyclonal antibody populations.

[0062] An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule comprising the antigen-binding portion (e.g., CDR) of an antibody, and is derived from that antibody. An antigen-binding molecule may comprise antigen-complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are

[0033] Another example of a suitable antigen-binding molecule is shown below. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to CD19. In a further embodiment, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen, comprising one or more complementarity-determining regions (CDRs) thereof. In a further embodiment, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule is an avimer. Comprises or consists of.

[0063] "Antigen" refers to any molecule capable of eliciting an immune response or being bound by an antibody or antigen-binding molecule. The immune response can involve either antibody production or the activation of specific immunologically competent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Additionally, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0064] The term "neutralize" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or alters the binding ability of the ligand by indirect means (such as altering the structure or energy of the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the bound protein from performing its biological function.

[0065] The term "autologous" refers to any material derived from the same individual that is to be later reintroduced. For example, the engineered autologous cell therapy (eACT™) described herein involves the collection of lymphocytes from a patient, which are then engineered, for example, to express a CAR construct and then administered back to the same patient.

[0066] The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species (eg, allogeneic T cell transplantation).

[0067] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, The vector may be a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentivirus vector, or any combination thereof.

[0068] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors. 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 are directed to the treatment of cancers, such as bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal lymphoma, and splenic lymphoma. Small intestine lymphoma (SMZL), esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, 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), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, The compositions may be used to reduce tumor size in tumors derived from rhodamine sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. In some embodiments, the cancer is multiple myeloma. Certain cancers may be responsive to chemotherapy or radiation therapy, while others may be refractory. Refractory cancer refers to cancer that is not amenable to surgical treatment, either because the cancer is initially unresponsive to chemotherapy or radiation therapy, or because the cancer becomes unresponsive over time.

[0069] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumor development, for example, a vaccine.

[0070] As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell and mediates a response in the second cell. As used herein, "cytokine" is meant to refer to a protein released by one cell population that acts on another cell as an intercellular mediator. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines include homeostatic cytokines, chemokines, and proinflammatory cytokines. , effector, and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote immune cell survival and proliferation, while proinflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF 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).

[0071] A "chemokine" is a type of cytokine that mediates cell chemotaxis or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0072] 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) contain binding domains that can interact with specific tumor antigens, allowing T cells to target and kill cancer cells that express the specific tumor antigen.

[0073] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability attributable to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled physician, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0074] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that constitutes a major component of the innate immune system. NK cells reject tumor and virus-infected cells. NK cells act by the process of apoptosis, or programmed cell death. NK cells are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (antibody-independent). Their T cell receptor (TCR) differentiates them from other types of lymphocytes. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4 + cell), cytotoxic T cell (TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8 + T cells (also known as killer T cells), memory T cells ((i) stem cell memory T cells like naive cells express CD45RO - , CCR7 + , CD45RA + , CD62L + (L-selectin), CD27 + , CD28 + and IL-7Rα +However, they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells; (ii) central memory TCM cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; and (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 + There are three types of T cells: regulatory T cells (TCR), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play the most important role in humoral immunity (antibody-mediated immunity). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and, after activation by antigen interaction, become memory B cells. In mammals, immature B cells are formed in the bone marrow, from which they are named.

[0075] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, the deletion of a coding or non-coding region, or portion thereof, or the insertion of a coding region, or portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell, which can be obtained from either a patient or a donor. The T cell can be modified to express a foreign construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the genome of the cell.

[0076] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced by any of these cells or the liver that result in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0077] The term "immunotherapy" refers to the treatment of a subject afflicted with a disease or at risk of developing a disease or suffering a recurrence of a disease by methods that involve inducing, enhancing, suppressing, or mitigating an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, tumor-infiltrating lymphocyte therapy, and the like. These treatments may include T-cell immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T-cell transplantation. However, those skilled in the art will recognize that the conditioning methods disclosed herein may enhance the efficacy of any transplanted T-cell therapy. Examples of T-cell therapies are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 7,741,465, U.S. Patent No. 6,319,494, U.S. Patent No. 5,728,388, and WO 2008 / 081035.

[0078] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be isolated from a subject using any of a number of techniques known to those skilled in the art, such as FICOLL™ isolation and / or apheresis. They may also be obtained from a unit of blood collected from the body. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0079] The term "engineered autologous cell therapy," also known as adoptive cell transfer and abbreviated as "eACT™," is the process of collecting a patient's own T cells and then genetically modifying them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. The T cells may express, for example, a chimeric antigen receptor (CAR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The CAR scFv can be designed, for example, to target CD19, a transmembrane protein expressed by cells in the B-cell lineage, including all normal B cells and B-cell malignancies, including, but not limited to, diffuse large B-cell lymphoma (DLBCL), unless otherwise specified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, NHL, CLL, and non-T-cell ALL. Examples of CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in their entireties.

[0080] As used herein, a "patient" includes any human afflicted with cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0081] As used herein, the term "in vitro cells" refers to any cells cultured ex vivo. In particular, in vitro cells can include T cells.

[0082] 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 may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the terms also refer to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0083] As used herein, "stimulation" refers to a primary response induced by binding of a stimulatory molecule with its cognate ligand, where the binding mediates a signal transduction event. A "stimulatory molecule" refers to a molecule on a T cell, e.g., a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, T cell activation, initiation of an immune response, proliferation, etc. Stimulatory 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.

[0084] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, such as, but not limited to, T cell proliferation and / or upregulation or downregulation of key molecules.

[0085] As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds a cognate costimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, T cell proliferation, activation, differentiation, etc. In addition to the primary signal, the signal provided by the T cell receptor (TCR) / CD3 complex is induced by stimulatory molecules, for example, by binding of the T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule. Costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, and herpes virus entry mediator (HVEM). entry mediator, human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transferase Examples of costimulatory ligands include immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed cell death (PD) L1. Costimulatory ligands include, but are not limited to, 4-1BB, B7-H3, a ligand that specifically binds to CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, and antibodies that specifically bind to costimulatory molecules present on T cells, such as lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0086] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, and a "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, C D3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LF A-1 (CD11a / CD18)), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations, young Or a combination thereof.

[0087] The terms "reducing" and "decreasing" are used interchangeably herein to refer to any change that is less than what is intended. "Decrease" and "reduce" are relative terms, requiring a comparison of a before and after measurement. "Decrease, reduce" and "reduce" include complete depletion.

[0088] "Treatment" of a subject or "treating" a subject " refers to any type of treatment or process performed on a subject, or the administration of an active ingredient to a subject, for the purpose of altering, mitigating, ameliorating, inhibiting, delaying, or preventing the onset, progression, manifestation, severity, or recurrence of symptoms, complications, or conditions, or biochemical markers associated with a disease. In some embodiments, "treatment" or "treating" includes partial remission. In other embodiments, "treatment" or "treating" includes complete remission.

[0089] Various aspects of the disclosure are described in further detail in the following subsections.

[0090] Characterization of the tumor microenvironment (TME) The present disclosure provides methods for characterizing the TME using gene expression profiling and / or intratumoral T cell density measurements prior to treatment with a chimeric receptor therapy (e.g., axicabtagene-ciloleucel (axi-cel)). As described herein, TME characteristics utilizing a predetermined gene set (e.g., Immunosign™ 21, Pan Cancer) and immune score (e.g., Immunosign™ 21) and / or intratumoral T cell density measurement or index (e.g., Immunoscore™) correlate with clinical outcomes of chimeric receptor therapy (e.g., axicabtagene-ciloleucel (axi-cel)).

[0091] A patient biopsy can be used as the starting material for analyzing the tumor microenvironment using gene expression profiling (e.g., digital gene expression using NanoString™). In some embodiments, the patient biopsy is obtained prior to treatment with a chimeric receptor therapy (e.g., axicabtagene-ciloleucel (axi-cel)).

[0092] Bioinformatics methods can be used to generate immune scores or scores to characterize the TME. In some embodiments, the immune score is a measure of immune-related genes that provides information on adaptive immunity, including T cell cytotoxicity, T cell differentiation, T cell attraction, and T cell adhesion, and immune suppression, including immune targeting, angiogenesis suppression, immune co-inhibition, and cancer stem cells. Bioinformatics methods can also include T cell-specific (effector T cell, Th1) genes, interferon pathway-related genes, chemokines, and immune checkpoints.

[0093] Expression profiling assays (e.g., the Immunosign™ clinical research assay utilizes nCounter™ technology (NanoString)) can be used to measure gene expression levels of multiple immune genes in a multiplex format. In some embodiments, cutoffs for high / low immune scores (e.g., Immunosign™ 21 scores) can be defined as the 25th percentile of scores observed across samples. In some embodiments, high scores indicate expression of immune-related genes potentially relevant to tumor response.

[0094] In some embodiments, the immune score is a measure of intratumoral T cell density. Intratumoral T cell density is determined, for example, by the CD3 T cell density in the tumor microenvironment. + T cells and / or CD8 +The abundance of T cells can be measured by detecting and quantifying T cells, such as T cells. For example, tumor biopsies can be sectioned and stained or labeled for T cell markers, such as CD3 and / or CD8, and the relative or absolute abundance of T cells can be quantified by a pathologist or measured using dedicated digital pathology software. In some embodiments, high A high / low Immunoscore (e.g., Immunoscore™) is assigned based on intratumoral T cell density. The high / low Immunoscore threshold can be defined, for example, as the median score observed across samples. In some embodiments, intratumoral T cell density is measured using flow cytometry and / or protein-based assays, such as Western blotting and ELISA.

[0095] Expression analysis and tumor-infiltrating T lymphocyte analysis and scoring can be used to examine the association between TME characteristics and response. In some embodiments, objective response (OR) is determined according to the revised IWG response criteria for malignant lymphoma (Cheson, 2007) and the IWG response criteria for malignant lymphoma (Cheson et al. Journal of Clinical Oncology 32, no. 27 (September 2014) 3059-3067). In some embodiments, the duration of response is assessed. In some embodiments, investigator-assessed progression-free survival (PFS) according to the Lugano response classification criteria is assessed.

[0096] Chimeric antigen receptors and T cell receptors Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be easily inserted into immune cells, including T cells, using techniques known in the art and expressed by the cells. With CARs, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. Chimeric antigen receptors can be made more potent by including a costimulatory (signaling) domain. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci. Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0097] In some embodiments, the costimulatory domain comprising a truncated hinge domain ("THD") further comprises some or all of a member of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or a fragment thereof.

[0098] In some embodiments, the THD is derived from a human complete hinge domain ("CHD"). In other embodiments, the THD is derived from a rodent, mouse, or primate (e.g., non-human primate) CHD of a costimulatory protein. In some embodiments, the THD is derived from a chimeric CHD of a costimulatory protein.

[0099] The costimulatory domain for a CAR or TCR of the present invention may further comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. The transmembrane domain may similarly be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to minimize interactions with other members of the receptor complex by avoiding binding of such domains to the transmembrane domain of the same or a different surface membrane protein. The transmembrane domain may be derived from either natural or synthetic sources. When natural in origin, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in the present invention include those derived from 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), C57 (SLAMF8), IL-1 ... D103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class I The antigen-binding protein may be derived from (i.e., comprise) a Ras1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof.

[0100] Optionally, a short linker may form the bond between any or some of the extracellular domain, transmembrane domain, and intracellular domain of the CAR. In some embodiments, the linker may be derived from glycine-glycine-glycine-glycine-serine repeats (G4S)n (SEQ ID NO:2) or GSTSGSGKPGSGEGSTKG (SEQ ID NO:1). In some embodiments, the linker has between 3 and 20 amino acids and an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSTSGSGKPGSGEGSTKG (SEQ ID NO:1).

[0101] The linkers described herein can also be used as peptide tags. The linker peptide sequence can be of any suitable length for linking one or more proteins of interest and is preferably designed to be sufficiently flexible to allow for proper folding and / or function and / or activity of one or both of the linked peptides. Thus, the linker peptide can have a length of 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acids. In some embodiments, the linker peptide can have a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids. In some embodiments, the linker has at least 7 and no more than 20 amino acids, at least 7 and no more than 19 amino acids, at least 7 and no more than 18 amino acids, at least 7 and no more than 17 amino acids, at least 7 and no more than 16 amino acids, at least 7 and no more than 15 amino acids, at least 7 and no more than 14 amino acids, at least 7 and no more than 13 amino acids, at least 7 and no more than 12 amino acids, or at least 7 and no more than 11 amino acids. In some embodiments, the linker has 15 to 17 amino acids, and in certain embodiments, 16 amino acids. In some embodiments, the linker has 10 to 20 amino acids. In some embodiments, the linker has 14 to 19 amino acids. In some embodiments, the linker has 15 to 17 amino acids. In some embodiments, the linker has 15 or 16 amino acids. In some embodiments, the linker has 16 amino acids. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0102] In some embodiments, a spacer domain is used. In some embodiments, the spacer domain is derived from CD4, CD8a, CD8b, CD28, CD28T, 4-1BB, or other molecules described herein. In some embodiments, the spacer domain may include a chemically derived dimerizer to control expression upon addition of a small molecule. In some embodiments, no spacer is used.

[0103] The intracellular (signaling) domain of the engineered T cells of the invention can transmit signals to the activation domain, which then activates at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including the secretion of cytokines.

[0104] In certain embodiments, suitable intracellular signaling domains include 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to LFA-1, CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC These include (i.e., comprise), but are not limited to, class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0105] In some embodiments, the chimeric antigen receptor (CAR) is axicabtagene ciloleucel (axi-cel). l) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy (Figure 1). Axi-cel (YESCARTA™) is approved by the US Food and Drug Administration for the treatment of patients with relapsed or refractory large B-cell lymphoma who have received two or more prior systemic therapies (Yescarta (axicabtagene-ciloleucel) (package insert). Santa Monica, CA: Kite Pharma; 2 017).

[0106] A TCR can be introduced to convey antigen reactivity. In some embodiments, antigen reactivity is restricted by MHC presentation of peptides. The TCR can be an α / β TCR, a γ / δ TCR, etc. In some embodiments, the TCR is an HPV-16 E7 TCR with mouse constant chains (2A binding). In some embodiments, the chains can be linked by an IRES or any 2A family member sequence (e.g., P2A, T2A, E2A, F2A, etc.). In some embodiments, the TCR is an HPV-recognizing TCR or other virus-reactive TCR (e.g., EBV, influenza, etc.). In some embodiments, a cancer-reactive or cancer-associated antigen-reactive TCR can be used (e.g., NYESO, MART1, gp100, etc.).

[0107] In some embodiments, the TCR is a normal / healthy peptide-reactive or other antigen-reactive / restricted TCR. In some embodiments, the TCR is reactive to mouse MHC or other non-human MHC. In some embodiments, the TCR is a class I-restricted TCR or a class II-restricted TCR.

[0108] antigen binding molecule Suitable CARs can be engineered by introducing an antigen-binding molecule that interacts with the target antigen (e.g., a cell surface antigen) to bind to the antigen. In some embodiments, the antigen-binding molecule is an antibody fragment thereof, such as one or more single-chain antibody fragments ("scFvs"). An scFv is a single-chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked to each other. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv maintains the ability of the parent antibody to specifically interact with the target antigen. An scFv is useful in chimeric antigen receptors because it can be engineered to be expressed as part of a single chain together with other CAR components. Id. 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 should be understood that the antigen-binding molecule is typically contained within the extracellular portion of the CAR so that it can recognize and bind to the antigen of interest. Bispecific and multispecific CARs having specificity for two or more targets of interest are considered within the scope of the present invention.

[0109] In some embodiments, the polynucleotide encodes a CAR or TCR comprising a THD of the present invention and an antigen binding molecule that specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is a tumor-associated surface antigen, such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, or CD4. , CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, pancreatic duct 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, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGF-1 receptor, IGF-II, IL-11R alpha, IL-13R -a2, influenza virus-specific antigen, CD38, insulin growth factor (IGFI)-1, intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa fever virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutated hsp70-2, mutated p53, mutated p53, mutated 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, surviving and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gp120), and any derivative or mutant of these surface markers.

[0110] Engineered T Cells and Uses The cells of the present disclosure can be obtained through T cells obtained from a subject. T cells may be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Additionally, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using numerous techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a buffer or medium appropriate for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, washing steps can be performed using semi-automated flow-through centrifuges, such as the Cobe™ 2991 cell processing device, Baxter CytoMate™, and the like. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions, with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0111] In some embodiments, T cells are isolated from PBMCs by depletion of monocytes, for example, by using lysis of red blood cells and centrifugation through a PERCOLL™ gradient. + , CD8 + , CD28 + , CD45RA + and CD45RO +Specific subpopulations of T cells, such as T cells from the negatively selected T cells, can be 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 accomplished by a combination of antibodies against surface markers unique to the cells being negatively selected. In some embodiments, cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells can be used. For example, negative selection can result in the deletion of CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-D. In some embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

[0112] In some embodiments, PBMCs are used directly for genetic modification with immune cells (such as CARs) using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated and sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion, for both cytotoxic and helper T lymphocytes.

[0113] In some embodiments, CD8 + These various CD8 cells + By identifying cell surface antigens associated with the cells, they are further sorted into naive cells, central memory cells, and effector cells. In some embodiments, expression of phenotypic markers of central memory T cells includes expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and are negative for granzyme B. In some embodiments, central memory T cells are negative for CD8 + , CD45RO + and CD62L +In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. + T cells are further sorted into subpopulations, e.g., CD4 + Helper T cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations that have cell surface antigens.

[0114] In some embodiments, immune cells, e.g., T cells, are isolated and then genetically modified using known methods, or the immune cells are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro prior to genetic modification. In another embodiment, immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR or TCR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514, and WO 2012 / 079000, the contents of which are incorporated herein by reference in their entireties. Typically, such methods involve contacting PBMCs or isolated T cells with stimulators and costimulators, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same beads serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads™ system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and WO 2012 / 129514, the contents of which are incorporated herein by reference in their entireties.

[0115] In some embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient afflicted with cancer or a tumor. In some embodiments, the donor subject is a human patient not afflicted with cancer or a tumor.

[0116] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient.

[0117] In some embodiments, the compositions are selected for parenteral delivery, for inhalation, or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of one of ordinary skill in the art. In some embodiments, a buffer is used to maintain the composition at physiological pH or a slightly lower pH, typically in the range of about 5 to about 8. In some embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water, in which the composition described herein, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, suitably stored. In some embodiments, the preparation involves formulating the desired molecule with a polymeric compound (such as polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, followed by delivery via depot injection. In some embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0118] In some embodiments, a method of treating cancer in a subject in need thereof includes T cell therapy. In some embodiments, the T cell therapy disclosed herein is an engineered autologous cell therapy (eACT™). According to this embodiment, the method may include collecting blood cells from the patient. The isolated blood cells (e.g., T cells) may then be engineered to express a CAR or TCR disclosed herein. In certain embodiments, the CAR T cells or TCR T cells are administered to the patient. In some embodiments, the CAR T cells or TCR T cells treat a tumor or cancer in the patient. In some embodiments, the CAR T cells or TCR T cells reduce the size of the tumor or cancer.

[0119] In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject who is not a patient.

[0120] In some embodiments, the T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells can be about 10 4 cells, approximately 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, or approximately 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×106 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 cells / kg.

[0121] In some embodiments, a therapeutically effective amount of CAR-positive viable T cells is a maximum dose of about 1 x 10 8 Up to approximately 1 x 10 CAR-positive viable T cells per kg of body weight 6 From about 2 x 10 6 Among these, CAR-positive surviving T cells are

[0122] Treatment method The methods disclosed herein can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor relapse, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

[0123] In some embodiments, the present disclosure provides a predictive tool for the clinical efficacy of T cell therapy by analyzing the tumor microenvironment pre-treatment.

[0124] The methods of the present invention can also be used in companion tests to provide information on whether an additional therapeutic agent, used in combination or sequentially, is more effective in subjects with certain tumor microenvironment characteristics. In some embodiments, the additional therapeutic agent can be a cytokine (e.g., IL-2, IL-15), a stimulatory antibody (e.g., anti-41BB, anti-OX-40), a checkpoint blockade therapy (e.g., CTLA4, PD-1), or an innate immune stimulator (e.g., TLR agonist, STING agonist). In some embodiments, the additional therapeutic agent can be a T cell-recruiting chemokine (e.g., CCL2, CCL1, CCL22, CCL17, and combinations thereof) and / or T cells. In some embodiments, the additional therapeutic agent(s) is administered systemically or intratumorally.

[0125] Cancers that can be treated include unvascularized, substantially unvascularized, or vascularized tumors. The cancer can also include solid or non-solid tumors. In some embodiments, the cancer is a cancer of the blood. In some embodiments, the cancer is a cancer of the white blood cells. In other embodiments, the cancer is a cancer of the plasma cells. In some embodiments, the cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute lymphocytic leukemia (ALL), and hemophagocytic lymphohistiocytosis (HLH)), B-cell prolymphocytic leukemia, B-cell acute lymphocytic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia (CML), chronic or acute granulomatous disease, chronic or acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, hemophagocytic syndrome (macrophage activation syndrome (MAS)), Hodgkin's disease, large cell granuloma (large cell granuloma), granuloma), leukocyte adhesion deficiency, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndromes (MDS), bone marrow disorders including, but not limited to, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (e.g., plasma dysplasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenstrom's macroglobulinemia, or a combination thereof.

[0126] 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.

[0127] In some embodiments, the method further comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent selected is a lymphodepleting (preconditioning) agent. Beneficial preconditioning treatment regimens, along with correlative beneficial biomarkers, are described in U.S. Provisional Patent Application Nos. 62 / 262,143 and 62 / 262,146. No. 2 / 167,750, which is incorporated herein by reference in its entirety. These include, for example, the administration of cyclophosphamide at the indicated beneficial dose (200 mg / m 2 / day~2000mg / m 2 / day) and the specified dose of fludarabine (20 mg / m 2 / day~900mg / m 2

[0010] Methods of conditioning a patient in need of T cell therapy are described, comprising administering to the patient about 500 mg / m2 / day of engineered T cells. One such dosing regimen comprises administering about 500 mg / m2 of engineered T cells to the patient prior to administration of a therapeutically effective amount of engineered T cells to the patient. 2 / day cyclophosphamide and approximately 60 mg / m 2 The method includes treating the patient by administering fludarabine / day to the patient daily for three days.

[0128] In some embodiments, the antigen binding molecule, transduced (or otherwise engineered) cell (such as a CAR or TCR), and chemotherapeutic agent are each administered in an amount effective to treat a disease or condition in a subject.

[0129] In some embodiments, compositions comprising immune effector cells expressing a CAR disclosed herein can be administered in conjunction with any number of chemotherapeutic agents, including alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolpropane. Ethyleneimine and methylamelamines, including trimethylolomelamine regimes; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterol Nitrogen mustards such as phenesterine, prednimustine, trofosfamide, and uracil mustard; carmustine, chlorozoline, Nimustine, toxin, fotemustine, lomustine, nimustine, ranimustine, etc. Torosourea; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calichiamat Isin, carabicin, carminomycin, carzinophilin, chromomycin cin, dactinomycin, daunorubicin, detorubicin, 6-diazo -5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, myco Phenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, Antibiotics such as zinostatin, zorubicin; methotrexate and 5-fluroxen Antimetabolites such as olauracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine Pyrimidine analogues such as uridine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotein, and trilostane; folic acid replacement fluids such as florinic acid; aceglatone; aldophosphamide Coside; aminolevulinic acid; amsacrine; bestrabucil; visan Tren; Edatraxate; Defofamine; Demecolcine; Diazicon; Elformithine; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenameth phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene) and Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In some embodiments, compositions comprising immune effector cells expressing a CAR and / or TCR disclosed herein are administered with antihormonal agents that act to modulate or inhibit the action of hormones on tumors, such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and thrombin. Antiestrogens, including remifen (Fareston), and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above, may also be administered in conjunction with appropriate chemotherapy combinations, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan™), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin™), and prednisone.

[0130] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week of administration of the engineered cells or nucleic acids. In other embodiments, the chemotherapeutic agent is administered one week to four weeks, one week to one month, one week to two months, one week to three months, one week to six months, one week to nine months, or one week to twelve months after administration of the engineered 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 comprises administering two or more chemotherapeutic agents.

[0131] A variety of additional therapeutic agents can be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO™), pembrolizumab (KEYTRUDA™), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche).

[0132] Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, but are not limited to, ibrutinib (IMBRUVICA™), ofatumumab (ARZERRA™), rituximab (RITUXAN™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), trastuzumab emtansine (KADCYLA™), imatinib (GLEEVEC™), cetethonib (CETECH ... Ximab (ERBITUX™), panitumumab (VECTIBIX™), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib , nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib mTOR inhibitors such as lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as the CDK inhibitor palbociclib.

[0133] In some embodiments, the composition comprising CAR immune cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and non-steroidal anti-inflammatory drugs (NSAIDs) including mycophenolate. Examples include: Representative NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Representative analgesics include acetaminophen, oxycodone, propoxyphene hydrochloride, tramadol. Representative glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors (e.g., TNF antagonists (e.g., etanercept (ENBREL™), adalimumab (HUMIRA™), and infliximab (REMICADE™))), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers also include monoclonal antibodies, as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0134] In some embodiments, the compositions described herein are administered in conjunction with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Among cytokines, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet growth factors; and transforming growth factors such as TGF-alpha and TGF-beta. Examples of cytokines include transforming growth factors (TGF); insulin-like growth factors I and II; erythropoietin (EPO, Epogen™, Procrit™); osteomorphic factors; interferons such as interferon-alpha, beta, and gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; interleukins (ILs) such as IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine refers to proteins derived from natural sources or from recombinant cell culture, and those derived from native sequence sites. Contains biologically active equivalents of cain.

[0135] Administration In some embodiments, the engineered T cells described herein are used to treat a malignant tumor in a patient in a method comprising: (a) obtaining a plurality of T cells having one or more chimeric receptors; and (b) administering an effective dose of the T cells to the patient.

[0136] In some embodiments, T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells can be about 10 4 cells, approximately 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, or approximately 10 8 In some embodiments, the CAR A therapeutically effective dose of T cells is approximately 2 x 10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 cells / kg.

[0137] In some embodiments, a therapeutically effective amount of CAR-positive viable T cells is a maximum dose of about 1 x 10 8 Up to approximately 1 x 10 CAR-positive viable T cells per kg of body weight 6 From about 2 x 10 6 Among these, CAR-positive surviving T cells are

[0138] monitoring In some embodiments, administration of the chimeric receptor T cell immunotherapeutic is performed in a licensed medical facility.

[0139] In some embodiments, the methods disclosed herein include monitoring a patient after administration of a chimeric receptor T cell immunotherapeutic (e.g., axicabtagene-ciloleucel (axi-cel)) at a licensed medical facility daily for at least seven days after infusion for signs and symptoms of CRS and neurotoxicity. In some embodiments, the patient is monitored daily for at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days.

[0140] In some embodiments, patients are instructed to remain within close proximity of a licensed medical facility for at least 4 weeks after infusion, hi some embodiments, patients are instructed to remain within close proximity of a licensed medical facility for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after infusion.

[0141] Clinical trials ZUMA-1 (NCT02348216) is a phase 1 / 2 multicenter, controlled trial of axi-cel in patients with refractory, aggressive large B-cell lymphoma (Figure 2) (Neelapu SN, Locke LF, et al. N Engl J Med. 2017;377:2531-2544). Axi-cel demonstrated a median survival time of 15.4 months. Responses were maintained over the median follow-up period. Of 108 patients with refractory large B-cell lymphoma treated with axi-cel in ZUMA-1 with a median follow-up period of 15.4 months, 82% had an objective response rate (ORR), with 58% experiencing an ORR. % achieved a complete response (CR) rate, with 42% experiencing durable responses, including 40% CRs. Cytokine release syndrome (CRS) and neurological events (NE) were reversible (13% had grade 3 or higher CRS, 28% had grade 3 or higher NE, and 3 had grade 5 adverse events).

[0142] Management of severe adverse reactions In some embodiments, the method includes managing an adverse reaction, in some embodiments, the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reaction, severe infection, cytopenia, and hypogammaglobulinemia.

[0143] In some embodiments, the signs and symptoms of the adverse reaction are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal 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.

[0144] Cytokine release syndrome In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. Patients experiencing Grade 2 or higher CRS (e.g., fluid-unresponsive hypotension or hypoxia requiring supplemental oxygen) should be monitored with continuous electrocardiogram telemetry and pulse oximetry. In some embodiments, patients experiencing severe CRS are considered for echocardiograms to assess cardiac function. Intensive care supportive therapy may be considered for severe or life-threatening CRS.

[0145] In some embodiments, the method includes monitoring the patient at a licensed medical facility for signs and symptoms of CRS daily for at least seven days after the infusion. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for four weeks after the infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention if any signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and a corticosteroid, as indicated, at the first sign of CRS.

[0146] Neurotoxicity In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients experiencing grade 2 or higher neurotoxicity should be monitored with continuous electrocardiogram telemetry and pulse oximetry. Intensive care supportive therapy is provided in the event of severe or life-threatening neurotoxicity.

[0147] In some embodiments, the method includes monitoring the patient at a licensed medical facility for signs and symptoms of neurotoxicity for at least seven days daily after the infusion, hi some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity for four weeks after the infusion.

[0148] Secondary malignancies In some embodiments, the patient is treated with CD19-directed genetically modified autologous T cell immunotherapy. Patients treated with the method may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies.

[0149] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that any definitions or terms set forth in a reference incorporated herein by reference differ from the terms and discussion set forth herein, the terms and definitions in this specification shall control.

[0150] Additional Embodiments One aspect of the present disclosure relates to a method of treating a malignant tumor, comprising measuring immune-related gene expression and / or T cell density (i.e., the tumor microenvironment) at one or more sites of the malignant tumor prior to administration (e.g., at least one infusion) of CAR-T cells or T cells expressing an exogenous TCR, in some embodiments, the measurements are performed prior to chemotherapeutic conditioning and administration of engineered T cells (e.g., CAR-T cells).

[0151] In some embodiments, the measurement involves determining a composite immune score based on immune-related gene expression, such as the ImmunoSign™ 21 or Immunosign™ 15 score. In some embodiments, the measurement involves determining a CD3 score, such as the Immunoscore™. + T cells and / or CD8 + and determining an immune score based on the intratumoral density of T cells, including T cells. In some embodiments, the measuring further comprises determining and assigning a relative score(s), such as high or low, based on a comparison of the subject's immune score(s) to a predetermined threshold. In some embodiments, such a predetermined threshold is determined or has been determined to have prognostic value with respect to treating a malignancy with the engineered T cells.

[0152] In some embodiments, the disclosed methods further comprise a step of optimizing treatment based on the measurement(s). For example, in some embodiments, the dose and / or schedule of administration of engineered T cells (e.g., CAR-T cells) is optimized based on the immune score(s) of the tumor microenvironment. In exemplary embodiments, subjects with low immune scores, such as low ImmunoSign™ 21 scores, are administered a higher dose of CAR-T cells than subjects with high immune scores. In some embodiments, subjects with low immune scores are administered about 25% higher, about 50% higher, or about 100% higher than subjects with high immune scores.

[0153] In additional and alternative exemplary embodiments, subjects with low immune scores receive one or more additional CAR-T cell infusions. In some embodiments, subjects with low pre-treatment immune scores are administered an initial dose of CAR-T cells, the treatment response is assessed, and if an incomplete response is observed, an additional TME immune score measurement step is performed. In some embodiments, if the subject has a high immune score after the initial dose, an additional dose of CAR-T cells is administered.

[0154] In some embodiments, the disclosed methods additionally or alternatively include a "pre-treatment" step in which subjects with low immune scores are treated with the intent of improving their immune scores prior to CAR-T administration. For example, in some embodiments, subjects with low immune scores are administered one or more immune stimulatory agents, such as cytokines, chemokines, or immune checkpoint inhibitors. In some embodiments, the additional measurement of the immune score is performed pre-treatment. It will be carried out.

[0155] In some embodiments, the prognostic value of a high Immunoscore for complete response to CAR-T therapy is considered when evaluating treatment options. For example, in some embodiments, subjects with a high Immunoscore receive CAR-T as an earlier line of treatment than subjects with a low Immunoscore.

[0156] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Example]

[0157] Example 1: Baseline TME immune gene expression as prognostic of axi-cel response Axicabtagene-ciloleucel (axi-cel; YESCARTA™) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy recently approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of patients with relapsed or refractory large B-cell lymphoma who have received two or more prior systemic therapies. The structure of the axi-cel CAR is illustrated in Figure 1.

[0158] ZUMA-1 (NCT02348216) is a phase 1 / 2 multicenter, controlled trial of axi-cel in patients with refractory, aggressive large B-cell lymphoma (Neelapu SN, Locke LF, et al. N Engl J Med. 2017;377:2531-2544). ZUMA-1 phases and cohort sizes are illustrated in Figure 2. Patients received both phase 2.0 × 10 6 CAR T cells / kg were administered.

[0159] Among 108 patients with refractory large B-cell lymphoma treated with axicabtagene-ciloleucel (axi-cel) in ZUMA-1, the overall response rate was 82%, with a 58% complete response (CR) rate. Cytokine release syndrome (CRS) and neurologic events (NE) were largely reversible (11% had grade 3 or higher CRS, 32% had grade 3 or higher NE, and four grade 5 adverse events (including two non-axi-cel-related events)). At a median follow-up of 15.4 months, the sustained response rate was 42%, including 40% CRs. At a median follow-up of 27.1 months, the sustained response rate was 39%, including 37% CRs.

[0160] A post-hoc analysis of the phase 2 ZUMA-1 study was designed to investigate the association of key pretreatment characteristics of the tumor immune microenvironment (TME). Baseline biopsies from 25 patients treated with axi-cel and axicabtagene-ciloleucel (axi-cel) with a minimum follow-up of 9 months were analyzed. Table 1 shows baseline patient and tumor characteristics and treatment outcomes.

[0161] [Table 1]

[0162] The objective response rate for these 25 patients was 80%, with 20 responders and 5 non-responders. At the subsequent data cutoff (minimum 12-month follow-up), one patient subsequently changed from a "non-responder" to a "responder" at 1 month.

[0163] As shown in Figure 3, tumor biopsies were taken at baseline and within 3 weeks of axi-cel administration. Baseline fresh-frozen core biopsies were analyzed with the Immunosign™ Clinical Research Assay using nCounter™ technology (NanoString) to measure gene expression levels of multiple immune genes in a multiplexed format. This assay was further developed to utilize minimal amounts of RNA from fresh-frozen or formalin-fixed tumor tissue. Predetermined bioinformatics methods and cutoffs were applied to immune-mediated tumor regression genes to identify adaptive immunity, including T cell cytotoxicity, T cell differentiation, T cell attraction, and T cell adhesion, as well as immune targeting and angiogenesis. Biosuppression, immune co-suppression, and immune suppression including cancer stem cells were evaluated (Immunosign™; Figure 4; Galon J, et al. Immunity. 2013;39:11-26; www.haliodx.com / clinical-research-services / immunosignr / ). Bioinformatics methods included T cell-specific (effector T cell, Th1) genes, interferon pathway-related genes, chemokines, and immune checkpoints. The cutoff for high / low Immunosign™ 21 score was defined as the 25th percentile of the scores observed across samples. High scores indicate expression of immune-related genes potentially relevant to tumor response.

[0164] Expression analysis and scoring were used to examine the association between TME features and response. A broader analysis using predefined gene sets was also applied, as shown in Table 2. A predefined 43-gene set consisting of Immusign™ 15, Immunosign™ 21, and other genes, and PanCancer Immune were used. All 763 genes from the Profiling Panel were included (nCounter PanCancer Immune Profiling Panel. https: / / www.nanostring.com / products / gene-expression-panels / hallmarks-cancer-gene-expression-panel-collection / pancancer-immune-profiling-panel). Exact probability tests and Wilcoxon signed rank tests with multiple testing correction by false discovery rate (Benjamini-Hochberg) were used.

[0165] [Table 2]

[0166] Pre-existing immune signatures of the tumor microenvironment (TME) correlated with response to axi-cel. As shown in Figure 5A, baseline Immunosign™ 21 scores of the TME were elevated in responders compared with nonresponders over a 9-month clinical follow-up period (P = 0.012). 85% (17 / 20) of responders had high Immunosign™ 21 scores, while 80% (4 / 5) of nonresponders had low Immunosign™ 21 scores (Figures 5B and 5C). In a sensitivity analysis including patients with delayed responses at 12 months, the association between Immunosign™ 21 and response was P = 0.053.

[0167] The top immune-related genes upregulated in the baseline TME in responders versus nonresponders included CTLA4, CD3g, CD3e, CD27, SH2B2, and ICOSL. Expression of other genes, including MHC class II genes and the cancer-testis antigens PRAME, MAGE, and SSX, was relatively decreased in the baseline TME in responders compared with nonresponders. The association with MHC class II genes is surprising, given the positive prognostic value of MHC class II expression in diffuse large B-cell lymphoma (Rimsza LM, et al. Blood. 2004;103:4251-4258). Additional genes differentially expressed in the TME in responders compared with nonresponders from the expanded 763-gene PanCancer Immune Profiling Panel are listed in Table 3.

[0168] [Table 3]

[0169] Example 2: Baseline intratumoral T cell density as a prognostic of axi-cel response Additional post-hoc analyses of ZUMA-1 in Phase 2 demonstrated key pre-treatment features of the tumor immune microenvironment (TME), particularly CD3 + T cell density and CD8 + This study was designed to investigate the association of T cell density and Immunoscore™ with response to axi-cel and immune gene expression (including Immunosign™ 21).

[0170] CD3 + T cells and CD8 + T cell density (cells / mm 2 Preconditioning tissue biopsies were formalin-fixed and paraffin-embedded (FFPE) for immunohistochemistry analysis. CD3 and CD8 staining was performed on two consecutive FFPE slices (4 μm) using a Benchmark™ XT station. The staining density of positive cells was measured using dedicated digital pathology software.

[0171] The Immunoscore™ assay measures CD8 + Cytotoxic T cells and CD3 + T cell density was measured and performed on formalin-fixed, paraffin-embedded tissue slides. Pre-treatment (before chemotherapeutic conditioning) tumor tissue biopsies were analyzed by immunohistochemistry to identify T cell subsets (CD3 + , CD8 + ) density (cells / mm 2 ) were analyzed. CD3 and CD8 staining was performed on two consecutive FFPE slices (4 μm) using a Benchmark™ XT station. Measurement of the area of ​​positive cells was performed using dedicated digital pathology software. For each subject, CD3 and CD8 immunohistochemical staining for 25 baseline biopsy samples was scored and converted to an ImmunoScore™ (a numerical index of T-cell density) using the HalioDx algorithm and analysis cutoffs described in Galon et al., J Pathol. 232:199-209 (2014). The median observed score was defined as the threshold for high and low Immunoscore™, where a high Immunoscore represents a relatively elevated intratumoral T-cell infiltration. Welch's t-test was used to compare the Immunoscore (quotient) between subjects showing CR. Target, CD3 and CD8 levels were compared in subjects with partial response, stable disease and progressive disease.

[0172] Results: Higher intratumoral CD3 + T cells and CD8 + The majority of patients with high T cell densities and high Immunoscore™ (all measured before treatment) achieved a CR (Figures 8A-8C). Overall, a higher pretreatment Immunoscore™ was associated with achieving a CR (P = 0.048; Figures 8A and 8B). CD3 + T cells and CD8 +Pretreatment intratumoral T cell density was positively associated with achieving CR (P = 0.025 and 0.049, respectively; Figures 9A and 9B). Patients who did not achieve CR mostly had low intratumoral CD3 + T cells and CD8 + showed a high density of T cells and a low Immunoscore™ (Figures 8A and 8C).

[0173] Immunoscore™ and Immunosign™ 21, assessed pretreatment on the same tumor biopsy, showed 82% agreement (95% CI, 65-93; r 2 = 0.451; Figure 10 ), suggesting a potential relationship between denser T cell infiltration and permissive gene signatures.

[0174] Findings from Examples 1 and 2 demonstrate the important role of the pre-treatment TME in response to CAR T cell therapy. Anti-CD19 CAR T therapy can overcome the poor prognosis associated with low Immunosign™ 21 scores or Immunoscore™.

[0175] Example 3: Tumor immune microenvironment Further analysis of patient samples revealed that CAR T cell proliferation occurred within two weeks of treatment and was accompanied by an increase in cytokines involved in the immune program. Pharmacodynamic profiling demonstrated a rapid increase above baseline in proliferation markers (IL-15 and IL-2), inflammatory markers (IL-6, CRP, SAA, IL-5, ferritin, IL-1Ra, IL-2Rα), immunoregulatory markers (GM-CSF, IFN-γ, IL-10), chemokines (IL-8, IP-10, MCP-1), and effector markers (granzyme B). Gene expression analysis suggested that pretreatment T cell-related genes (CD3ε, CD28, and CTLA4), innate immunity-related genes (MX1, ISG15, and MYD88), and B cell-related genes (CD19, CD79B, and PAX5) may correlate with clinical outcome (Figure 11).

[0176] CAR T cells were detected in the TME of Zuma-1 patients (7 to 21 days post-treatment) using in-situ hybridization (ISH) for CAR T RNA and immunohistochemistry (IHC) with antibodies recognizing distinct CAR epitopes described in WO 2018 / 013563 and WO 2018 / 053790. An increased rate of sustained response at 1 year was observed in patients with reduced pre-treatment tumor burden. These data suggest that engraftment of CAR T cells commensurate with tumor burden can overcome large tumors. CAR T cell therapy was associated with changes in the TME. Analysis of transcripts from a pre-defined panel of 43 immune genes showed upregulation in tumors 7 to 21 days post-treatment. In response to treatment, increases in immune inhibitory checkpoints (PD-L1, CTLA4, LAG3, TNFRSF18, ICOS), IFN-related genes (IRF1, STAT1, STAT4, IFNγ) and chemokines (CXCL9, CCL2, CCL5), effector genes (CD8A, GNLY, GZMA, GZMM, GZMB, GZMH), and the proliferation marker IL-15 were observed (Figure 12). Nanostring analysis demonstrated elevated expression of the IL-15 and PD-L1 genes in subjects with complete or partial responses (Figures 13A and 13B).

[0177] Biopsies from patients with large cell lymphoma and B-cell acute lymphoblastic leukemia (B-ALL) were evaluated for CD19 and PD-L1 expression. At least one-third of subjects who relapsed after Axi-cel had tumors negative for CD19 expression. Analysis of B-cell and immune-related molecules at progression revealed negative CD19 expression. + Tumor cells or CD19 - Recurrence was confirmed by tumor cells. At baseline, 94% (16 / 17) of evaluable patients were CD19 positive. Post-progression tumor biopsies from 21 evaluable patients showed that 33% were CD19 negative and 62% were PD-L1 positive.

Claims

1. 1. A method of treating a malignant tumor in a patient, comprising: (a) analyzing a tumor biopsy from said patient to characterize the tumor microenvironment; (b) administering to said patient an effective dose of T cells comprising one or more chimeric receptors; wherein the effective dose is determined using characteristics of the tumor microenvironment.

2. 10. The method of claim 1, wherein the tumor microenvironment is characterized using gene expression profiling, intratumoral T cell density measurement, or a combination thereof.

3. 3. The method of claim 2, wherein the gene expression profiling comprises measuring the expression levels of a specific panel of genes.

4. 4. The method of any one of claims 1 to 3, comprising measuring the expression level of one or more genes selected from CD3G, STAT4, CD3E, CD3D, GZMK, GZMM, PRF1, CD8A, ICOS, CXCL10, STAT1, IL15, CCR2, CCL2, IRF1, TBX21, GZMA, CXCR3, GZMB, CD69, CXCL11, and combinations thereof.

5. 5. The method of any one of claims 1 to 4, comprising measuring the expression level of one or more genes selected from CTLA4, GZMH, CD8A, PDCD1, CD3G, IRF1, CX3CL1, TNFRSF9, CD3E, GZMA, CXCL10, TSLP, REN, GZMB, TNFRSF18, CCL2, GZMK, CXCL11, CD69, CD247, CCL5, STAT4, CD274, GNLY, ITGAE, LAG3, IL15, LTK, PRF1, CD3D, PF4, TBX21, ICOS, CXCL9, IFNG, VEGFA, STAT1, GZMM, CXCL13, CXCR3, CCR2, IL17A, PROM1, and combinations thereof.

6. The method of any one of claims 1 to 5, comprising measuring the expression levels of genes selected from the PanCancer Immune Profiling Panel.

7. The method according to any one of claims 1 to 6, comprising measuring the expression level of a B cell marker.

8. The method according to any one of claims 1 to 7, comprising measuring the expression level of a T cell marker.

9. The method of any one of claims 1 to 8, comprising measuring the expression levels of a specific gene panel comprising genes associated with the innate immune response.

10. The method of claim 9, wherein the specific gene panel comprises markers of cytotoxic cells, dendritic cells, macrophages, or granulocytes.

11. The method of any one of claims 1 to 10, further comprising determining an immune score based on the gene expression profile and / or the intratumoral T cell density.

12. 12. The method of claim 11, further comprising adjusting the total dose using the immune score.

13. The effective dose is at least 1 x 10 per kg of body weight ６ CAR-positive viable T cells The method according to any one of claims 1 to 12, comprising:

14. The method of any one of claims 1 to 13, wherein the chimeric receptor is a chimeric antigen receptor (CAR).

15. The method of any one of claims 1 to 14, wherein the chimeric receptor is a T cell receptor (TCR).

16. The malignant tumors include solid tumors, sarcomas, carcinomas, lymphomas, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, and the like. lymphoma (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, one or more of B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma 16. The method of any one of claims 1 to 15, wherein the disease is selected from the group consisting of myeloma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., dysplasmocyte proliferation, 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), or a combination thereof.

17. The method of any one of claims 1 to 16, wherein the effective dose is optimized to increase the likelihood that the patient will respond to chimeric receptor therapy.

18. 1. A method for determining whether a patient will respond to chimeric receptor therapy, comprising: (a) analyzing a tumor biopsy from said patient to characterize the tumor microenvironment using a gene expression profile; (b) determining an immune score based on the gene expression profile; and (c) determining whether the patient will respond to chimeric receptor therapy based on the immune score; and A method comprising:

19. 1. A method for determining whether a patient will respond to chimeric receptor therapy, comprising: (a) obtaining a tumor biopsy from the patient prior to treatment; (b) analyzing the tumor biopsy to characterize the tumor microenvironment; (c) determining whether the patient will respond to chimeric receptor therapy based on the characteristics of the tumor microenvironment; and A method comprising:

20. 1. A method of treating a malignant tumor in a patient, comprising: (a) analyzing a tumor biopsy from said patient prior to chimeric receptor treatment to characterize the tumor microenvironment; (b) determining whether the patient will respond to chimeric receptor therapy based on the characteristics of the tumor microenvironment; (c) administering to said patient an effective dose of T cells comprising one or more chimeric receptors; wherein the effective dose is determined using characteristics of the tumor microenvironment.

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