Chimeric receptor t cell therapy using characteristics of tumor microenvironment

Abstract

The present disclosure provides chimeric receptor T cell therapy using the characteristics of the tumor microenvironment. The present disclosure provides methods of treating a malignant tumor comprising administering an effective dose of a chimeric receptor (e.g., CAR or TCR) genetically modified T cell immunotherapy. Some aspects of the present disclosure relate to methods of characterizing a tumor microenvironment prior to infusion and determining an effective dose of T cell immunotherapy.

Description

[0001] This application is a divisional application based on a patent application with an application date of April 12, 2019, a priority date of April 12, 2018, an application number of 201980025411.7, and an invention name of “Chimeric receptor T cell therapy using characteristics of the tumor microenvironment”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 656,825, filed on April 12, 2018; U.S. Provisional Patent Application No. 62 / 827,770, filed on April 1, 2019; and U.S. Provisional Patent Application No. 62 / 831,946, filed on April 10, 2019, which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 11, 2019, is named K-1065_01_SL.txt, and is 8KB in size. Technical Field

[0006] The present disclosure specifically relates to chimeric receptor T cell therapy using characteristics of the tumor microenvironment. Background of the Invention

[0008] Human cancers, by their very nature, consist of normal cells that have undergone a genetic or epigenetic transformation into abnormal cancer cells. At this point, cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells such as T and B lymphocytes from successfully targeting cancer cells.

[0009] Human T cell therapies rely on enriched or modified human T cells to target and kill a patient's cancer cells. 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 capable of interacting with specific tumor antigens, enable T cells to target and kill cancer cells that express specific tumor antigens. SUMMARY OF THE INVENTION

[0011] Characteristics of the tumor microenvironment (TME) prior to infusion of CAR T cells can affect clinical outcomes. The present disclosure provides methods for evaluating the TME using immune-related gene expression signatures of the TME and / or intratumoral T cell density to correlate with outcomes.

[0012] The various aspects and embodiments described below may be combined unless the context clearly indicates otherwise.

[0013] In one aspect, the present disclosure provides a method for treating a malignant tumor in a patient, 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 characteristics of the tumor microenvironment.

[0014] 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.

[0015] In some embodiments, gene expression profiling includes determining the expression level of a specified group of genes. In some embodiments, the group includes CD3G, STAT4, CD3E, CD3D, GZMK, GZMM, PRF1, CD8A, ICOS, CXCL10, STAT1, IL15, CCR2, CCL2, IRF1, TBX21, GZMA, CXCR3, GZMB, CD69, CXCL11, and combinations thereof.

[0016] In some embodiments, the group 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. In some embodiments, the group includes a Pan-Cancer Immune Profiling Panel.

[0017] In some embodiments, the method comprises determining the expression level of a B cell marker. In some embodiments, the B cell marker is selected from BLK, CD19, CR2, MS4A1, TNFRSF17 and a combination thereof.

[0018] In some embodiments, the method comprises determining the expression level of a T cell marker. In some embodiments, the T cell marker is selected from CD2, CD2E, CD3G, CD6 and a combination thereof.

[0019] In some embodiments, the method comprises determining the expression level of a specified set of genes, the specified set of genes comprising genes associated with an innate immune response. In some embodiments, the specified set of genes comprises markers for cytotoxic cells, dendritic cells, macrophages, granulocytes, and combinations thereof.

[0020] In some embodiments, the specified set of genes includes genes selected from CD8, BLC2, and combinations thereof.

[0021] In some embodiments, the designated group of genes includes genes selected from CCL12, CCL17, and combinations thereof.

[0022] In some embodiments, the designated group of genes includes genes selected from APOE, CCL7, and combinations thereof.

[0023] In some embodiments, the designated group of genes includes genes selected from the group consisting of CMA1, CSF3R, and combinations thereof.

[0024] In some embodiments, the method comprises determining the expression level of one or more genes selected from CTLA4, CD3g, CD3e, CD27, SH2B2, ICOSL, and combinations thereof.

[0025] In some embodiments, the method comprises determining 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.

[0026] In some embodiments, the method comprises determining the expression level of one or more genes selected from CD27, SH2B2, ICOSLG, and combinations thereof.

[0027] In some embodiments, the method comprises determining 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.

[0028] In some embodiments, the method includes determining the density of T cells in the tissue microenvironment (i.e., T cell density within the tumor), for example, by immunohistochemical staining of a tumor biopsy. In some embodiments, the method includes determining the density of CD3+ and / or CD8+ T cells in the tissue microenvironment.

[0029] In some embodiments, the methods include determining an immune score based on a gene expression profile.

[0030] In some embodiments, the method includes determining an immune score based on intratumoral T cell density. In some embodiments, the method includes using the immune score to adjust the total dose of CAR-T cells. In some embodiments, the method includes providing an immunomodulatory compound or interference to increase the immune score before administering the CAR-T cells.

[0031] In some embodiments, an effective dose comprises at least 1×10 6 CAR-positive live T cells.

[0032] In some embodiments, the chimeric receptor targets a tumor antigen. In some embodiments, the chimeric receptor targets a tumor antigen selected from a tumor-associated surface antigen, such as 5T4, alpha-fetoprotein (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, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialylganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipids, 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, IGFI receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific antigen;CD38, insulin growth factor (IGF1), intestinal carboxylesterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, mesothelin, MN-CA IX, MUC-1, mut hsp70 -2, mutant p53, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, protease, 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, survival and telomerase, TAG-72, extra domain A (EDA) and extra domain B of fibronectin and Al domain of tenascin C (TnC Al), 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 derivatives or variants of these surface markers. ;

[0033] In some embodiments, the chimeric receptor specifically targets CD19.

[0034] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the chimeric receptor is a T cell receptor (TCR).

[0035] In some embodiments, the malignancy is one or more of a solid tumor, a sarcoma, a carcinoma, a lymphoma, 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 myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), T cell lymphoma, B cell acute lymphoid leukemia ("BALL"), T cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, 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, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasma cell neoplasms (e.g., plasma cell dyscrasias, 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.

[0036] In some embodiments, the malignancy is diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBC), high-grade B-cell lymphoma, non-Hodgkin's lymphoma, metastatic melanoma, transformed follicular lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma.

[0037] In some embodiments, the effective dose is optimized to increase the likelihood that the patient will respond to treatment.

[0038] In some embodiments, a tumor biopsy is obtained from a patient prior to treatment with the chimeric receptor therapy.

[0039] In one aspect, the present disclosure includes a method of determining whether a patient will respond to a chimeric receptor therapy, comprising: (a) analyzing a tumor biopsy from the patient using gene expression profiling to characterize the tumor microenvironment; (b) determining an immune score based on the gene expression profile; and (c) determining whether the patient will respond to the chimeric receptor therapy based on the immune score.

[0040] In one aspect, the present disclosure includes a method for determining whether a patient will respond to a chimeric receptor therapy, comprising: (a) analyzing a tumor biopsy from the patient by quantifying intratumoral T cell (e.g., CD3+ and / or CD8+ T cell) density to characterize the tumor microenvironment; (b) determining an immune score based on the intratumoral T cell density; and (c) determining whether the patient will respond to the chimeric receptor therapy based on the immune score.

[0041] In one aspect, the present disclosure provides a method for treating a malignant tumor in a patient, 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 the 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 microenvironmental characteristics of the tumor.

[0042] In some embodiments, the method further comprises using the characteristics of the tumor microenvironment to determine whether other therapies will improve clinical outcomes. In some embodiments, chimeric receptor therapy is administered with other therapies. In some embodiments, other therapies are administered in conjunction with chimeric receptor therapy. In some embodiments, other therapies are administered before or after chimeric receptor therapy.

[0043] In some embodiments, the method further comprises administering cytokine therapy. In some embodiments, the cytokine is IL-2 or IL-15.

[0044] In some embodiments, the method further comprises administering a stimulatory antibody. In some embodiments, the stimulatory antibody is anti-41BB or OX-40.

[0045] In some embodiments, the method further comprises administering a checkpoint blockade therapy. In some embodiments, the checkpoint blockade therapy comprises CTLA4 or PD-1.

[0046] In some embodiments, the method further comprises administering an innate immune stimulator. In some embodiments, the innate immune stimulator comprises a TLR or STING agonist.

[0047] In some embodiments, gene expression profiling includes determining the expression level of proliferation markers, inflammatory markers, immunomodulatory markers, effectors and / or chemokines. In some embodiments, the method includes determining the expression level 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 includes determining the expression level of one or more genes selected from GM-CSF, IFN-γ, IL-10, and combinations thereof. In some embodiments, the method includes determining the expression level of one or more genes selected from IL-8, IP-10, MCP-1, and combinations thereof. In some embodiments, the method includes determining the expression level of granzyme B. In some embodiments, the method includes determining the expression level of CD3ε, CD28, and CTLA4. In some embodiments, the method includes determining the expression level of MX1, ISG15, and MYD88. In some embodiments, the method includes determining the expression level of CD19, CD79B, and PAX5. In some embodiments, the method includes determining the expression level of PD-L1 and / or CD19. In some embodiments, a tumor biopsy is obtained prior to treatment with CAR T cell therapy. In some embodiments, a 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, a tumor biopsy is obtained 7 days, 14 days, 21 days, or 28 days after treatment with CAR T cell therapy.

[0048] The present disclosure includes the following embodiments:

[0049] 1. A method of treating a malignant tumor in a patient, comprising:

[0050] (a) analyzing a tumor biopsy from the patient to characterize the tumor microenvironment; and

[0051] (b) administering to the 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.

[0052] 2. The method of embodiment 1, wherein the tumor microenvironment is characterized using gene expression profiling, intratumoral T cell density measurement, or a combination thereof.

[0053] 3. The method of embodiment 2, wherein the gene expression profiling comprises determining the expression levels of a specified group of genes.

[0054] 4. The method of any of the preceding embodiments, comprising determining the expression level of one or more genes selected from the group consisting 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.

[0055] 5. The method of any of the preceding embodiments, comprising determining the expression level of one or more genes selected from the group consisting of 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.

[0056] 6. The method of any one of the preceding embodiments, comprising determining the expression level of a gene selected from the Pan Cancer Immune Profiling Panel.

[0057] 7. The method according to any one of the preceding embodiments, comprising determining the expression level of a B cell marker.

[0058] 8. The method according to any one of the preceding embodiments, comprising determining the expression level of a T cell marker.

[0059] 9. The method of any one of the preceding embodiments, comprising determining the expression level of a specified group of genes, the specified group of genes comprising genes associated with an innate immune response.

[0060] 10. The method of embodiment 9, wherein the specified group of genes includes markers of cytotoxic cells, dendritic cells, macrophages or granulocytes.

[0061] 11. The method according to any one of the preceding embodiments, further comprising determining an immune score based on gene expression profile and / or intratumoral T cell density.

[0062] 12. The method of embodiment 11, further comprising using the immune score to adjust the total dose.

[0063] 13. The method of any of the preceding embodiments, wherein the effective dose comprises at least 1×10 6 CAR-positive live T cells per kg body weight.

[0064] 14. The method according to any one of the preceding embodiments, wherein the chimeric receptor is a chimeric antigen receptor (CAR).

[0065] 15. The method according to any one of the preceding embodiments, wherein the chimeric receptor is a T cell receptor (TCR).

[0066] 16. The method of any of the preceding embodiments, wherein the malignancy is one or more of a solid tumor, a sarcoma, a carcinoma, a lymphoma, 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 myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), T cell lymphoma, B cell acute lymphoid leukemia ("BALL"), T cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid Dendritic cell neoplasms, 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, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasma cell neoplasms (e.g., plasma cell dyscrasias, 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.

[0067] 17. The method according to any one of the preceding embodiments, wherein the effective dose is optimized to increase the likelihood that the patient will respond to chimeric receptor therapy.

[0068] 18. A method of determining whether a patient will respond to a chimeric receptor therapy, comprising:

[0069] (a) analyzing a tumor biopsy from the patient using gene expression profiling to characterize the tumor microenvironment;

[0070] (b) determining an immune score based on the gene expression profile; and

[0071] (c) determining whether the patient will respond to chimeric receptor therapy based on the immune score.

[0072] 19. A method of determining whether a patient will respond to a chimeric receptor therapy, comprising:

[0073] (a) obtaining a tumor biopsy from the patient prior to treatment;

[0074] (b) analyzing the tumor biopsy to characterize the tumor microenvironment; and

[0075] (c) determining whether the patient will respond to chimeric receptor therapy based on the characteristics of the tumor microenvironment.

[0076] 20. A method of treating a malignant tumor in a patient, comprising:

[0077] (a) analyzing tumor biopsies from patients prior to chimeric receptor therapy to characterize the tumor microenvironment;

[0078] (b) determining whether the patient will respond to chimeric receptor therapy based on characteristics of the tumor microenvironment; and

[0079] (c) administering to the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The drawings are for illustration purposes only and are not limiting.

[0082] Figure 1 A schematic diagram of the chimeric antigen receptor (CAR) construct configuration is shown.

[0083] Figure 2 Schematic diagram of the ZUMA-1 clinical trial showing patients receiving axicabtagene ciloleucel. AE, adverse event; axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; CR, complete response; CRS, cytokine release syndrome; DLBCL, diffuse large B-cell lymphoma; NE, neurologic event; ORR, objective response rate; PMBCL, primary mediastinal B-cell lymphoma; TFL, transformed follicular lymphoma.

[0084] Figure 3A schematic diagram of the ZUMA-1 clinical trial protocol and paired biopsy schedule is shown.

[0085] Figure 4 Demonstrated for evaluation of key immune pathways within the tumor microenvironment Clinical Research Analysis Group.

[0086] Figure 5A show 21 score and clinical outcomes in a sample of 25 patients treated with axicabtagene ciloleucel (axi-cel) with at least 9 months of follow-up. One patient subsequently converted from a "non-responder" to a "responder" at 12 months of follow-up. Figure 5B Shows the high and low baselines 21 The proportion of patients who responded to axi-cel therapy. Figure 5C Shows the high and low baselines 21 Score Proportion of patients who do not respond to axi-cel therapy. High / Low The 21 score cutoff was defined as the 25th percentile of scores observed in the sample. Figure 5B and Figure 5C The dot in the middle indicates low 21 ratings.

[0087] Figure 6 In samples from 25 patients who received at least 25 months of axicabtagene ciloleucel (axi-cel) treatment, responders were compared with nonresponders. The expression of 21 genes was differentially expressed.

[0088] 7A to 7C The top three immune environment genes elevated in tumors from responders are shown: CTLA4 ( Fig. 7A ), CD3γ( Figure 7B ) and CD3ε( Figure 7C ) from a prespecified 43-panel immune gene panel of 25 patients treated with axicabtagene ciloleucel (axi-cel) and followed for at least nine months.

[0089] Fig. 8A show Association between mean percentiles and clinical outcomes in samples from treated patients. Figure 8B Shows a high baseline Proportion of clinical outcomes observed in patients. Figure 8C Shows a low baseline The proportion of patients for whom the clinical outcome was observed. High / Low 21 The score cutoff was defined as the median score observed in the sample. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.

[0090] Fig. 9A and Fig. 9B Shown are the densities of CD3+ cells and CD8+ cells, respectively, in baseline biopsies of treated patients, complete responders and patients demonstrating something other than a complete response.

[0091] Fig.10 Plotted to assess baseline biopsies from treated patients 21 ratings and The correlation between them.

[0092] Fig.11 Gene expression analysis 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) in the TME before treatment and clinical outcomes at 1-year follow-up after axi-cel treatment are shown. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.

[0093] Fig.12 Figure 3 shows the fold changes in gene expression in the TME 7 to 21 days after CAR-T treatment, showing relative changes compared to baseline for immunosuppressive 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 the proliferation marker IL-15.

[0094] Fig.13A and Fig. 13B IL-15 ( Fig.13A ) and PD-L1( Fig. 13B ) Nanostring analysis of differential changes. Pre-Tx: before treatment; W1-2: Weeks 1-2; W4: Week 4. DETAILED DESCRIPTION OF THE INVENTION

[0096] The present disclosure relates to methods for treating malignant tumors in patients using characteristics of the tumor microenvironment of patient biopsies. The present disclosure is based in part on the surprising discovery that the characteristics of the tumor microenvironment of patient biopsies obtained before chimeric receptor treatment can be used to prognose clinical outcomes. As described herein, the tumor microenvironment profile before chimeric receptor treatment is used to determine an effective dose to affect the clinical outcomes of chimeric receptor (e.g., CAR or TCR) T cell therapy.

[0097] definition

[0098] In order to make the present invention more easily understood, some terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0099] 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.

[0100] As used herein, the term "or" should be construed as inclusive and encompasses both "or" and "and," unless expressly stated or obvious from the context.

[0101] As used herein, the term "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0102] As used herein, the terms "such as" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed in the specification.

[0103] 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 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 4, 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. Also included are any larger numbers or fractions therebetween.

[0104] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller number or fractions therebetween.

[0105] 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 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 4, 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. Also included are any larger numbers or fractions therebetween.

[0106] Throughout the specification, the words "comprising" or variations (comprising / comprises) will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. It should be understood that wherever aspects are described herein with the language "comprising", other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0107] Unless explicitly stated or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within or above 1 standard deviation as practiced in the art. "About" or "approximately" can mean a range of up to 10% (i.e., ± 10%). Therefore, "about" can be understood as being greater than or less than the value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001%. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. In addition, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to 5 times. When specific values ​​or compositions are provided in the present disclosure, unless otherwise stated, it should be assumed that "about" or "approximately" means within an acceptable error range for the specific value or composition.

[0108] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers).

[0109] The units, prefixes, and symbols used herein are provided in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2 nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5 th ed., (2013), Academic Press; and "The Oxford DictionaryOf Biochemistry And Molecular Biology", Cammack et al. eds., 2 nd ed, (2006), Oxford University Press provides those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0111] "Administering" refers to the physical introduction of a medicament into a subject using any of various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of preparations 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" means the mode of administration except enteral and topical administration, usually by injection and including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, via non-parenteral routes, such as oral administration of preparations. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or local. It can also be, for example, once, repeatedly, and / or administered in one or more extended periods.

[0112] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into highly variable regions, called complementary determining regions (CDRs), which are interspersed with more conservative regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

[0113] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugated antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations.

[0114] "Antigen binding molecule", "antigen binding portion" or "antibody fragment" refers to any molecule comprising an antigen binding portion (e.g., CDR) of an antibody derived from the molecule. Antigen binding molecules may include antigen complementary 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 peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, antigen binding molecules bind to antigens on tumor cells. In some embodiments, antigen binding molecules bind to antigens on cells involved in hyperproliferative diseases or bind to viral or bacterial antigens. In certain embodiments, antigen binding molecules bind to BCMA, CLL-1 or FLT3. In further embodiments, antigen binding molecules are antibody fragments that specifically bind to antigens, including one or more complementary determining regions (CDRs). In further embodiments, antigen binding molecules are single-chain variable fragments (scFv). In some embodiments, the antigen binding molecule comprises or consists of an avimer.

[0115] "Antigen" refers to any molecule that triggers an immune response or can be bound by an antibody or antigen binding molecule. The immune response may involve the production of antibodies or the activation of specific immune cells, or both. It will be readily appreciated by those skilled in the art that any macromolecule (including almost all proteins or peptides) 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 certain tissue (e.g., cancer cells), or can be widely expressed. In addition, fragments of larger molecules can serve as antigens. In some embodiments, the antigen is a tumor antigen.

[0116] The term "neutralization" 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, an antigen binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand, or changes the binding capacity of the ligand by indirect means (such as structural or energy changes in the ligand). In some embodiments, an antigen binding molecule, scFv, antibody, or fragment thereof prevents the protein bound thereto from performing a biological function.

[0117] The term "autologous" refers to any material that originates from the same individual and is subsequently reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein TM ) method involves collecting lymphocytes from a patient, subsequently engineering them to express, for example, a CAR construct, and then administering them to the same patient.

[0118] The term "allogeneic" refers to any material derived from one individual that is subsequently introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0119] The terms "transduction" and "transduced" refer to the process of introducing foreign DNA into cells by viral vectors (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0120] "Cancer" refers to a large and diverse group 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, which invade adjacent tissues and may also metastasize to distant sites of the body via the lymphatic system or blood. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods disclosed herein can be used to reduce the size of a tumor originating from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, 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 zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer , adrenal cancer, soft tissue sarcoma, urethral cancer, liver cancer penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in children, lymphocytic lymphoma, bladder cancer, cancer kidney or ureter, renal pelvic cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancer (including asbestos induced cancer), other B cell malignancies, and combinations of the above cancers. In some embodiments, the cancer is multiple myeloma. Specific cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer is cancer that cannot be treated surgically and that does not respond initially to chemotherapy or radiation therapy, or becomes unresponsive over time.

[0121] As used herein, "anti-tumor effect" refers to a biological effect that can be presented as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various tumor-related physiological symptoms. Anti-tumor effect can also refer to the prevention of tumor occurrence, such as vaccines.

[0122] As used herein, "cytokine" refers to a non-antibody protein released by a cell response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. As used herein, cytokines refer to proteins released by a cell population that act on another cell as an intercellular mediator. Cytokines can be expressed endogenously by cells or applied to subjects. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to propagate immune responses. Cytokines can induce various responses in receptor cells. Cytokines can include steady-state cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, steady-state cytokines (including interleukins (IL) 7 and IL-15) promote immune cell survival and proliferation, and proinflammatory cytokines can promote inflammatory responses. Examples of steady-state 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) γ. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF) 2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0123] "Chemokine" is a class of cytokines that mediate cell chemotaxis or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), γ-induced protein 10 (IP-10), and thymus activation-regulated chemokine (TARC or CCL17).

[0124] As used herein, "chimeric receptors" refer to engineered surface-expressed molecules that can recognize specific molecules. 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.

[0125] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., engineered CAR T cells) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, such as during clinical trials in human subjects, in animal model systems used to predict efficacy in humans, or by measuring the activity of an agent in an in vitro assay.

[0126] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells or B cells. NK cells are a type of cytotoxic (cytotoxic / cell toxic) lymphocytes that represent the main components of the innate immune system. NK cells reject tumors and virus-infected cells. It works through the process of apoptosis or programmed cell death. Because it does not require activation to kill cells, it is called "natural killer". T cells play a major role in cell-mediated immunity (without antibody participation). Its T cell receptor (TCR) distinguishes itself from other lymphocyte types. The thymus (specialized organ of the immune system) is primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell-like memory TSC cells (e.g., naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3 and LFA-1, and display many unique functional properties of memory cells; (ii) central memory TCM cells express L-selectin and CCR7, they secrete IL-2, but not IFNγ or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7, but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKT) and γδT cells. On the other hand, B cells play a major role in humoral immunity (with the participation of antibodies). They produce antibodies and antigens, perform the role of antigen presenting cells (APCs), and become memory B cells after activation through antigen interaction. In mammals, immature B cells are formed in the bone marrow, which is where the name of B cells comes from.

[0127] The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte (e.g., a T cell), which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) incorporated into the genome of the cell.

[0128] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Ab, cytokines, and complement) that results in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues in the body of a vertebrate.

[0129] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting a disease or recurrence of a disease, which treatment is performed by methods including inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT) TM ) and allogeneic T cell transplantation. However, those skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Publications No. 2014 / 0154228 and No. 2002 / 0006409, U.S. Patent No. 7,741,465, U.S. Patent No. 6,319,494, U.S. Patent No. 5,728,388, and International Patent Application Publication No. WO 2008 / 081035.

[0130] The T cells of immunotherapy can be from any source known in the art. For example, T cells can be differentiated from a hematopoietic stem cell population in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. Many techniques known to those skilled in the art can also be used, such as FICOLL TM Isolation and / or apheresis, obtaining T cells from a unit of blood collected from a subject. Other methods of isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0131] The term "engineered autologous cell therapy" (which may be abbreviated as "eACT TM", also known as adoptive cell transfer) is the process of collecting a patient's own T cells and subsequently genetically altering them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a specific tumor antigen, which is linked to an intracellular signaling portion comprising at least one co-stimulatory domain and at least one activation domain. CAR scFv can be designed to target, for example, CD19, which is 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), primary mediastinal large B cell lymphoma, high-grade B cell lymphoma, and DLBCL caused by follicular lymphoma, NHL, CLL and non-T cell ALL unless otherwise specified. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 227237, 2014 / 0099309, and 2014 / 0050708, the entireties of which are incorporated by reference.

[0132] As used herein, "patient" includes any person suffering from cancer (eg, lymphoma or leukemia).Herein, the terms "subject" and "patient" are used interchangeably.

[0133] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, the in vitro cell may include a T cell.

[0134] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, which have many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0135] As used herein, "stimulation" refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. "Stimulatory molecules" are molecules on T cells, such as T cell receptors (TCR) / CD3 complexes that specifically bind to cognate stimulatory ligands present on antigen presenting cells. "Stimulatory ligands" are ligands that can specifically bind to stimulatory molecules on T cells when present on antigen presenting cells (e.g., APCs, dendritic cells, B cells, etc.), thereby mediating the primary response of T cells (including but not limited to activation, initiation of immune response, proliferation, etc.). Stimulatory ligands include but are not limited to anti-CD3 antibodies, MHC class I molecules loaded with peptides, super agonist anti-CD2 antibodies, and super agonist anti-CD28 antibodies.

[0136] As used herein, a "co-stimulatory signal" refers to a signal that, when combined with a primary signal (eg, TCR / CD3 ligation), results in a T cell response (eg, but not limited to, proliferation and / or up- or down-regulation of key molecules).

[0137] As used herein, "costimulatory ligands" include molecules on antigen presenting cells (APCs) that specifically bind to cognate costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.). Costimulatory ligands induce signals in addition to the primary signals provided by stimulatory molecules (e.g., provided by the binding of T cell receptor (TCR) / CD3 complexes to major histocompatibility complex (MHC) molecules loaded with peptides). Co-stimulatory ligands may include, but are not limited to, 3 / TR6, 4-1BB ligands, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible co-stimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD) L1. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0138] "Co-stimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as but not limited to proliferation. Co-stimulatory molecules include but are not limited to 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45. CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD1 9a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (α; β; δ; ε; γ; ζ), CD30, CD37 , CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96(Tactile), CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

[0139] The terms "reduce" and "reduce" are used interchangeably herein and refer to any change from the original. "Reduce" and "reduce" are relative terms requiring a comparison between before and after the measurement. "Reduce" and "reduce" include complete consumption.

[0140] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on the subject, or the administration of an active agent to the subject, in order to achieve the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with the disease. In some embodiments, "treatment" or "treatment" includes a partial response. In another embodiment, "treatment" or "treatment" includes a complete response.

[0141] Various aspects of the invention are described in further detail in the following subsections.

[0142] Characterization of the Tumor Microenvironment (TME)

[0143] 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, using a pre-defined gene set (e.g., 21, Pan Cancer) and immune scores (e.g., 21) and / or intratumoral T cell density measurements or indicators (e.g., ) correlate TME characteristics with the clinical efficacy of chimeric receptor therapies, such as axicabtagene ciloleucel (axi-cel).

[0144] Patient biopsies can be used for gene expression profiling (e.g., using NanoString TM In some embodiments, the patient biopsy is obtained prior to treatment with a chimeric receptor therapy (e.g., axicabtagene ciloleucel (axi-cel)).

[0145] Bioinformatics methods can be used to generate one or more immune scores to characterize the TME. In some embodiments, the immune score is a measure of immune-related genes that provide information about acquired immunity, including T cell cytotoxicity, T cell differentiation, T cell attraction, T cell adhesion, and immunosuppression, including immune orientation, angiogenesis inhibition, immune co-suppression and cancer stem cells. Bioinformatics methods can also include T cell-specific (effector T cells, Th1) genes, interferon pathway-related genes, chemokines, and immune checkpoints.

[0146] Expression profiling methods (e.g. Clinical Research Analysis Method Technology (NanoString) can be used to measure the gene expression levels of multiple immune genes in a multiplex format. In some embodiments, high / low immune scores (e.g. 21 score) can be defined as the 25th percentile of the scores observed in the sample. In some embodiments, a high score indicates the expression of immune-related genes that are potentially related to tumor response.

[0147] In some embodiments, the immune score is a measure of intratumoral T cell density. Intratumoral T cell density can be determined, for example, by detecting and quantifying T cells such as CD3+ T cells and / or CD8+ T cells in the tumor microenvironment. For example, a tumor biopsy can be sliced, stained or labeled with T cell markers (e.g., CD3 and / or CD8), and the relative or absolute abundance of T cells can be quantified by a pathologist or determined using dedicated digital pathology software. In some embodiments, a high / low immune score (e.g., ). A high / low immune score threshold can be defined as, for example, the median score observed in the sample. In some embodiments, flow cytometry and / or protein-based assays such as Western blot and ELISA are used to determine intratumoral T cell density.

[0148] Expression and tumor infiltrating T lymphocyte analysis and scoring can be used to examine the association between TME features and responses. In some embodiments, objective response (OR) is determined according to the revised IWG malignant lymphoma response criteria (Cheson, 2007) and determined by IWG malignant lymphoma response criteria (Cheson et al., Journal of Clinical Oncology 32, No.27 (September 2014) 3059-3067). In some embodiments, duration is assessed. In some embodiments, progression-free survival (PFS) assessed by researchers according to Lugano response classification criteria is assessed.

[0149] Chimeric Antigen Receptor and T Cell Receptor

[0150] Chimeric antigen receptor (CAR or CAR-T) is a genetically engineered receptor. According to techniques known in the art, these engineered receptors can be easily inserted into immune cells including T cells and expressed by them. Using CAR, a single receptor can be programmed to recognize a specific antigen, and when bound to the antigen, activate immune cells to attack and destroy cells carrying the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. Chimeric antigen receptors incorporate costimulatory (signal transduction) domains to increase their effectiveness. See U.S. Pat. Nos. 7,741,465 and 6,319,494, and 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).

[0151] In some embodiments, the costimulatory domain including a truncated hinge domain ("THD") also comprises some or all of a member of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

[0152] In some embodiments, the THD is derived from a human complete hinge domain ("CHD"). In other embodiments, the THD is derived from a costimulatory protein of a rodent, murine, or primate (e.g., non-human primate) CHD. In some embodiments, the THD is derived from a chimeric CHD of a costimulatory protein.

[0153] The co-stimulatory domain of the CAR or TCR of the present invention may further include a transmembrane domain and / or an intracellular signal transduction domain. The transmembrane domain may be designed to be fused with the extracellular domain of CAR. Similarly, it may be fused with the intracellular domain of CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex. The transmembrane domain may be derived from a natural source or a synthetic source. If the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein.The transmembrane region specifically used in the present invention can be derived from (i.e., include) 4-1BB / CD137, NK cell activating receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, C EACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, with CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen 1 (LFA-1; CD11a / CD18), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death protein 1 (PD-1), PSGL1, SELPLG (CD162), signal transducing lymphocyte activation molecules (SLAM proteins), 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 fragments, truncations or combinations thereof.

[0154] Optionally, a short linker can form a connection between any or some extracellular, transmembrane and intracellular domains of CAR. In some embodiments, the linker can be derived from a repeating sequence of glycine-glycine-glycine-glycine-serine (G4S) n (SEQ ID NO: 2) or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1). In some embodiments, the linker comprises 3-20 amino acids, and the amino acid sequence is 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).

[0155] Joints as described herein can also be used as peptide tags.Joint peptide sequences can have any suitable length to connect one or more target proteins, and are preferably designed to have enough flexibility, so as to allow the appropriate folding and / or function and / or activity of one or two peptides connected thereto.Therefore, the length of the joint peptide can be no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acids.In some embodiments, the length of the joint peptide can be 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 3 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids. In some embodiments, the linker comprises at least 7 and no more than 20 amino acids, at least 7 and no more than 19 amino acids, at least 7 and no more than 18 amino acids, at least 7 and no more than 17 amino acids, at least 7 and no more than 16 amino acids, at least 7 and no more than 15 amino acids, at least 7 and no more than 14 amino acids, at least 7 and no more than 13 amino acids, at least 7 and no more than less than 12 amino acids or at least 7 and no more than 11 amino acids. In certain embodiments, the linker comprises 15-17 amino acids, and in specific embodiments, comprises 16 amino acids. In some embodiments, the linker comprises 10-20 amino acids. In some embodiments, the linker comprises 14-19 amino acids. In some embodiments, the linker comprises 15-17 amino acids. In some embodiments, the linker comprises 15-16 amino acids. In some embodiments, the linker comprises 16 amino acids. In some embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0156] In some embodiments, a spacer is used. In some embodiments, the spacer is derived from CD4, CD8a, CD8b, CD28, CD28T, 4-1BB or other molecules described herein. In some embodiments, the spacer may include a chemically induced dimer to control expression when a small molecule is added. In some embodiments, a spacer is not used.

[0157] The intracellular (signal transduction) domain of the engineered T cell of the present invention can provide a signal to the activation domain, and then the activation domain activates at least one normal effector function of the immune cell. For example, the effector function of the T cell can be cytolytic activity or auxiliary activity, including the secretion of cytokines.

[0158] In certain embodiments, suitable intracellular signal transduction domains include (i.e., comprise), but are not limited to 4-1BB / CD137, NK cell activating receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a , CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (I COS), 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), Ly108), lymphocyte function-associated antigen 1 (LFA-1; CD11a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death protein 1 (PD-1), PSGL1, SELPLG (CD162), signal transducing 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.

[0159] In some embodiments, the chimeric antigen receptor (CAR) is axicabtagene ciloleucel (axi-cel). Axicabtagene ciloleucel (axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy ( Figure 1 ). Axicabtagene ciloleucel(axi-cel)(YESCARTA TM Yescarta (axicabtagene ciloleucel) [package insert]. Santa Monica, CA: Kite Pharma; 2017) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with relapsed or refractory large B-cell lymphoma who have received ≥ 2 prior systemic therapies.

[0160] TCR can be introduced to transfer antigen reactivity. In some embodiments, antigen reactivity is limited by MHC presentation of peptides. TCR can be α / βTCR, γ / δTCR, etc. In some embodiments, TCR is HPV-16E7TCR (2A connected) with murine constant chain. In some embodiments, the chain can be connected by IRES or the sequence of any 2A family member (e.g., P2A, T2A, E2A, F2A, etc.). In some embodiments, TCR is HPV that recognizes TCR, or other virus reactive TCR (e.g., EBV, influenza, etc.). In some embodiments, cancer or cancer-associated antigen reactive TCR (e.g., NYESO, MART1, gp100, etc.) can be used.

[0161] 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 murine or other non-human MHC. In some embodiments, the TCR is a class I or class II restricted TCR.

[0162] Antigen Binding Molecules

[0163] Suitable CARs can be engineered to bind antigens (e.g., cell surface antigens) by incorporating antigen binding molecules that interact with their targeted antigens. In some embodiments, the antigen binding molecule is an antibody fragment thereof, such as one or more single-chain antibody fragments ("scFv"). ScFv is a single-chain antibody fragment having heavy and light chain variable regions of antibodies linked together. See U.S. Patents Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. ScFv retains the ability of the parent antibody to specifically interact with the target antigen. ScFv is useful in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain together with other CAR components. Same as above. 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 appreciated that the antigen binding molecule is generally contained in the extracellular portion of the CAR, enabling it to recognize and bind to an antigen of interest. Bispecific and multispecific CARs with specificity for more than one target of interest are contemplated within the scope of the present invention.

[0164] In some embodiments, the polynucleotide encodes a CAR or TCR comprising a THD of the 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 selected from tumor-associated surface antigens such as 5T4, alpha-fetoprotein (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, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, asialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipids, 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, IGFI receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific antigen;CD38, insulin growth factor (IGF1), intestinal carboxylesterase, kappa chain, LAGA-la, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules with tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, muthsp70-2, mutant p53, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, protease, 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, survival and telomerase, TAG-72, extra domain A (EDA) and extra domain B of fibronectin and Al domain of tenascin C (TnCA1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor 2 (VEGFR2), virus-specific surface antigens such as HIV-specific antigens (such as HIV gpl20), and any derivatives or variants of these surface markers.;

[0165] Engineered T cells and their applications

[0166] The cells of the present disclosure can be obtained by obtaining T cells from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained using many techniques known to those skilled in the art (e.g., FICOLL TM The cells are collected from a blood unit collected from a subject by separation and / or apheresis. In certain embodiments, the cells collected by apheresis are washed to remove the plasma portion and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. It is understood that the cells can be separated, for example, by using a semi-automatic non-reverse flow centrifuge (e.g., Cobe TM 2991 Cell Processor, Baxter CytoMate TMIn some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other saline solutions with or without buffers. In certain embodiments, undesirable components are removed from the aliquot sample. Other methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0167] In some embodiments, the monocytes are depleted by lysing the erythrocytes and depleting the monocytes (e.g., via PERCOLL TM Gradients, by separation using centrifugation) to separate T cells from PBMCs. In some embodiments, specific subsets of T cells, such as CD4 + 、CD8 + 、CD28 + 、CD45RA + and CD45RO + T cells. For example, enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies against surface markers specific to the negatively selected cells. In some embodiments, cell sorting and / or sorting via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells can be used. For example, to enrich CD4 + Cells, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20 and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

[0168] In some embodiments, PBMC is directly used for the genetic modification (such as CAR) of the immune cell using the method as described herein.In certain embodiments, after separating PBMC, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes are sorted into initial T cells, memory T cells and effector T cell subsets before or after genetic modification and / or amplification.

[0169] In some embodiments, by identifying + CD8 + The cells are further sorted into naive cells, central memory cells, and effector cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is granzyme B negative. In some embodiments, central memory T cells are CD8+ 、CD45RO + and CD62L + In some embodiments, the effector T cells are negative for CCR7, CD28, CD62L, and CD127 and positive for granzyme B and perforin. In certain embodiments, the CD4 + T cells can be further sorted into subsets. For example, CD4 + T helper cells are sorted into naive cells, central memory cells, and effector cells.

[0170] In some embodiments, immune cells such as T cells are genetically modified using known methods after separation, or immune cells are activated and amplified in vitro (or, differentiated in the case of progenitor cells) before being genetically modified. In another embodiment, immune cells such as T cells are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding CAR or TCR), and then activated and / or amplified in vitro. Methods for activating and amplifying T cells are known in the art and described in, for example, U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby incorporated by reference as a whole. In general, such methods include contacting PBMCs or separated T cells with stimulants and co-stimulators (e.g., anti-CD3 and anti-CD28 antibodies, typically adhered to beads or other surfaces) in a culture medium with appropriate cytokines (e.g., IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads act as "surrogate" antigen presenting cells (APCs). An example is System, CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, feeder cells and appropriate antibodies and cytokines are used to activate T cells and stimulate them to proliferate, using methods described in, for example, U.S. Pat. Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514 (the contents of which are hereby incorporated by reference in their entirety).

[0171] In certain embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In some embodiments, the donor subject is a human patient who does not suffer from cancer or a tumor.

[0172] 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.

[0173] In some embodiments, the composition is selected for parenteral delivery, inhalation or delivery through the digestive tract, such as oral. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In some embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8. In some embodiments, when parenteral administration is considered, the composition is in the form of a pyrogen-free parenteral acceptable aqueous solution containing the composition described herein in a pharmaceutically acceptable vehicle, with or without other therapeutic agents. In some embodiments, the carrier for parenteral injection is sterile distilled water, wherein the composition described herein, with or without at least one other therapeutic agent is formulated as a sterile isotonic solution, and appropriately preserved. In some embodiments, the preparation comprises a formulation of the desired molecule with a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads or liposomes to provide controlled or sustained release of the product, and then delivered via a reservoir injection. In some embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0174] In some embodiments, a method of treating cancer in a subject in need thereof comprises T cell therapy. In some embodiments, the T cell therapy disclosed herein is an engineered autologous cell therapy (eACT TM ). According to this embodiment, the method may include collecting blood cells from the patient. Then, the separated blood cells (e.g., T cells) can be engineered to express the CAR or TCR disclosed herein. In a specific embodiment, 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 tumors or cancers in the patient. In some embodiments, the CAR T cells or TCR T cells reduce the size of the tumor or cancer.

[0175] In some embodiments, the donor T cells used in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, the donor T cells used in T cell therapy are obtained from a non-patient subject.

[0176] 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 is about 10 4cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In some embodiments, the therapeutically effective amount of CAR T cells is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or about 9×10 7 cells / kg.

[0177] In some embodiments, the therapeutically effective amount of CAR-positive live T cells is about 1×10 6 about 2×10 6 CAR-positive live T cells, the maximum dose is about 1×10 8 CAR-positive live T cells.

[0178] Treatment

[0179] 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 a tumor from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0180] In some embodiments, the present invention provides a prognostic tool for the clinical efficacy of T cell therapy by analyzing the tumor microenvironment before treatment.

[0181] The method of the present invention can also be used for accompanying testing to inform whether other therapies will be more effective in combination or sequential use in subjects with certain tumor microenvironment characteristics. In some embodiments, other therapies can be cytokines (e.g., IL-2, IL-15), stimulating antibodies (e.g., anti-41BB, OX-40), checkpoint blockade (e.g., CTLA4, PD-1) or natural immune stimulants (e.g., TLR, STING agonists). In some embodiments, other therapies can be chemokines (e.g., CCL2, CCL1, CCL22, CCL17, and combinations thereof) and / or T cells that recruit T cells. In some embodiments, one or more other therapies are administered systemically or intratumorally.

[0182] Cancers that can be treated include tumors that are not vascularized, not substantially vascularized, or vascularized. Cancers can also include solid tumors or non-solid tumors. In some embodiments, the cancer is a hematological cancer. 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 lymphoid leukemia (ALL) and hemophagocytic lymphohistiocytosis (HLH), B cell prolymphocytic leukemia, B cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, 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, leukocyte adhesion defect, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome (MDS), myeloid diseases including but not limited to acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, plasma cell neoplasms (e.g., plasma cell dyscrasias; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytomas), POEMS syndrome (Crow-Fukase syndrome; Takatsuki disease; PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom's macroglobulinemia, or a combination of the above.

[0183] 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.

[0184] In some embodiments, the method further includes administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent selected is a lymphodepletion (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens and related beneficial biomarkers are described in U.S. Provisional Patent Application Nos. 62 / 262,143 and 62 / 167,750, which are hereby incorporated by reference in their entirety. These describe methods for conditioning patients who need T cell therapy, including administering a specified beneficial dose of cyclophosphamide (200 mg / m 2 / day-2000mg / m 2 / day) and the specified dose of fludarabine (20 mg / m 2 / day-900mg / m 2 One such dosage regimen involves treating a patient comprising administering to the patient about 500 mg / m 2 / day of cyclophosphamide and about 60 mg / m 2 / day of fludarabine for 3 days, and then the patient is administered a therapeutically effective amount of engineered T cells.

[0185] In some embodiments, the antigen binding molecule, transduced (or otherwise engineered) cell (e.g., CAR or TCR), and chemotherapeutic agent are each administered in an amount effective to treat a disease or disorder in a subject.

[0186] In certain embodiments, the compositions comprising the immune effector cells expressing CAR disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN TM); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine resume; nitrogen mustards, such as phenoxyethanol, anthracene, and phenoxyethanol. mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate , pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; and aminolevulinic acid. acid; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; podophyllinic acid; acid; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL TM , Bristol-Myers Squibb) and doxetaxel ( Rhone-Poulenc Rorer; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C ; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as Targretin TM (bexarotene), Panretin TM , (alitretinoin); ONTAK TM(denileukindiftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, a composition comprising CAR and / or TCR expressing immune effector cells can be administered in combination with an anti-hormone agent, the anti-hormone agent acting to regulate or inhibit the effect of hormones on tumors, such as anti-estrogen, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. Where appropriate, a combination of chemotherapeutic agents is also administered, including but not limited to CHOP, i.e. cyclophosphamide Doxorubicin (hydroxydoxorubicin), vincristine and prednisone.

[0187] In some embodiments, the chemotherapeutic agent is administered within one week of or after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month before the administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0188] A variety of other therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful other therapeutic agents include PD-1 inhibitors, such as nivolumab, ), pembrolizumab, ), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche).

[0189] Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, but are not limited to, ibrutinib Ofatumumab Rituximab Bevacizumab Trastuzumab trastuzumabemtansine Imatinib Cetuximab Panitumumab 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 toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib Trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitors (palbociclib).

[0190] In some embodiments, the composition comprising CAR immune cells is administered together with an anti-inflammatory agent.Anti-inflammatory agents or drugs may include but are not limited to steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists, (e.g., etanercept Adalimumab and infliximab Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecule. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0191] In some embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-β; platelet growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO, ); osteoinductive factors; interferons such as interferon-α, β and -γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines.

[0192] Application

[0193] In some embodiments, the engineered T cells described herein are used to treat a malignancy in a patient in the following method: (a) obtaining a plurality of T cells comprising one or more chimeric receptors; (b) administering an effective dose of the T cells to the patient.

[0194] 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 9cells, or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In some embodiments, the therapeutically effective amount of CART cells is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg, or about 9×10 7 cells / kg.

[0195] In some embodiments, the therapeutically effective amount of CAR-positive live T cells is about 1×10 6 About 2×10 6 CAR-positive live T cells, up to about 1×10 8 The maximum dose of viable CAR-positive T cells.

[0196] monitor

[0197] In some embodiments, administration of chimeric receptor T cell immunotherapy is performed at a certified medical facility.

[0198] In some embodiments, the methods disclosed herein include monitoring the patient for signs and symptoms of CRS and neurotoxicity daily at a certified medical facility for at least 7 days after administration of a chimeric receptor T cell immunotherapy (such as axicabtagene ciloleucel (axi-cel)) following infusion. In some embodiments, the patient is monitored daily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0199] In some embodiments, the patient is instructed to stay near a certified medical facility for at least 4 weeks after the infusion. In some embodiments, the patient is instructed to stay near a certified medical facility for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the infusion.

[0200] Clinical trials

[0201] ZUMA-1 (NCT02348216) is a phase 1 / 2 multicenter pivotal study of axicabtagene ciloleucel (axi-cel) in patients with refractory aggressive large B-cell lymphoma. Figure 2 )(Neelapu SN, Locke LF, et al. NEngl J Med. 2017; 377: 2531-2544). Axicabtagene ciloleucel (axi-cel) maintained sustained responses at a median follow-up of 15.4 months. In ZUMA-1, 82% of 108 patients with refractory large B-cell lymphoma treated with axicabtagene ciloleucel (axi-cel) with a median follow-up of 15.4 months showed an objective response rate (ORR), 58% complete response (CR), and 42% sustained responses, including 40% CR. Cytokine release syndrome (CRS) and neurological events (NE) were reversible (13% ≥ grade 3 CRS, 28% ≥ grade 3 NE: 3 grade 5 adverse events).

[0202] Management of serious adverse reactions

[0203] In some embodiments, the method includes the management of adverse reactions. In some embodiments, the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, serious infections, cytopenias, and hypogammaglobulinemia.

[0204] In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, including arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, allergic reactions, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

[0205] Cytokine Release Syndrome

[0206] In some embodiments, the method includes identifying CRS based on clinical manifestations. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. Patients experiencing ≥ grade 2 CRS (e.g., hypotension, hypoxia that is unresponsive to fluids, or requiring supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients experiencing severe CRS, consider performing an echocardiogram to evaluate cardiac function. For severe or life-threatening CRS, intensive care supportive therapy may be considered.

[0207] In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS every day in a certified medical institution after infusion for at least 7 days. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS 4 weeks after infusion. In some embodiments, the method includes: if signs or symptoms of CRS occur at any time, the patient is advised to seek medical attention immediately. In some embodiments, the method includes supportive treatment with tocilizumab or tocilizumab and corticosteroids at the first sign of CRS.

[0208] Neurotoxicity

[0209] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients experiencing ≥ Grade 2 neurotoxicity should undergo continuous cardiac telemetry and pulse oximetry monitoring. Provide intensive care supportive therapy for severe or life-threatening neurotoxicity.

[0210] In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity daily at a certified medical facility for at least 7 days after infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity within 4 weeks after infusion.

[0211] Secondary malignant tumors

[0212] In some embodiments, patients treated with CD19-directed, genetically modified autologous T cell immunotherapy may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies.

[0213] All publications, patents and patent applications mentioned in this specification are incorporated herein 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 references cited herein should not be construed as an admission that such references are prior art to the present invention. To the extent that any definitions or terms provided in the references incorporated by reference differ from the terms and discussions provided herein, the terms and definitions of the present invention shall prevail.

[0214] Other Implementations

[0215] The present disclosure relates to a method for treating a malignant tumor on the one hand, comprising measuring immune-related gene expression and / or T cell density at one or more sites (i.e., tumor microenvironment) of a malignant tumor before administering (e.g., at least one infusion) CAR-T cells or T cells expressing exogenous TCRs. In some embodiments, the measurement is performed before administering chemotherapy-regulated and engineered T cells (e.g., CAR-T cells).

[0216] In some embodiments, the measuring comprises determining a comprehensive immune score based on immune-related gene expression, e.g. 21 or 15 Scoring. In some embodiments, the measuring comprises determining an immune score based on the intratumoral density of T cells, including CD3+ and / or CD8+ T cells, e.g. In some embodiments, the measuring further comprises determining and assigning a relative score, such as high or low, based on a comparison of the subject's immune score with a predetermined threshold. In some embodiments, determining or having determined that such a predetermined threshold has prognostic value for treating a malignancy with engineered T cells.

[0217] In some embodiments, the methods disclosed herein further include a step of optimizing treatment based on the measurements. 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 of the tumor microenvironment. In an exemplary embodiment, a subject with a low immune score (e.g., a low In some embodiments, a subject with a low immune score is administered a higher dose of CAR-T cells than a subject with a high immune score. In some embodiments, a subject with a low immune score is administered a dose that is about 25% higher, or about 50% higher, or about 100% higher than a subject with a high immune score.

[0218] In other and alternative exemplary embodiments, subjects with low immune scores receive one or more other CAR-T cell infusions. In some embodiments, a first dose of CAR-T cells is administered to a subject with a low immune score before treatment, 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's immune score is higher after the first administration, an additional administration of CAR-T cells is performed.

[0219] In some embodiments, the disclosed methods additionally or alternatively include a "pre-treatment" step, in which a subject with a low immune score is treated with the goal of improving its immune score before CAR-T administration. For example, in some embodiments, one or more immunostimulants, such as cytokines, chemokines, or immune checkpoint inhibitors, are administered to a patient with a low immune score. In some embodiments, additional measurements of the immune score are performed prior to treatment.

[0220] In some embodiments, the prognostic value of a high immune score for a complete response to a CAR-T-based therapy should be considered when evaluating treatment options. For example, in some embodiments, subjects with a high immune score receive CAR-T administration as an earlier route of therapy than subjects with a low immune score.

[0221] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all references cited in this application are expressly incorporated herein by reference. Example

[0222] Example 1: Baseline TME immune gene expression as prognostic of axi-cel response

[0223] Axicabtagene ciloleucel(axi-cel; ) is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy currently approved by the US FDA and EMA for the treatment of patients with relapsed or refractory large B-cell lymphoma who have previously undergone 2 or more systemic therapies. Figure 1 The structure of axi-cel CAR is schematically depicted.

[0224] ZUMA-1 (NCT02348216) is a phase 1 / 2 multicenter pivotal study of axicabtagene ciloleucel (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). The phase and population size of ZUMA-1 are Figure 2 Patients received 2.0×10 6 CAR T cells / kg.

[0225] In ZUMA-1, 108 patients with refractory large B-cell lymphoma treated with axicabtagene ciloleucel (axi-cel) achieved an overall response rate of 82% and a complete response (CR) rate of 58%. Cytokine release syndrome (CRS) and neurologic events (NE) were mostly reversible (11% ≥ grade 3 CRS; 32% ≥ grade 3 NE; 4 grade 5 adverse events (including 2 non-axi-cel related events). At a median follow-up of 15.4 months, the sustained response rate was 42%, including 40% CR. At a median follow-up of 27.1 months, the sustained response rate was 39%, including 37% CR.

[0226] Phase 2 post hoc analysis of ZUMA-1 was designed to explore associations with key features of the tumor immune microenvironment (TME) prior to treatment. Baseline biopsies from 25 patients treated with axicabtagene ciloleucel (axi-cel) and followed for at least 9 months were analyzed. Table 1 shows patient and tumor baseline characteristics and treatment outcomes.

[0227] Table 1. Patient and Tumor Baseline Characteristics and Treatment

[0228]

[0229]

[0230] The objective response rate for these 25 patients was 80%, with 20 responders and 5 non-responders. At the subsequent data cutoff (minimum 12 months follow-up), 1 patient subsequently converted from a "non-responder" to a "responder" at month 1.

[0231] like Figure 3 Tumor biopsies were obtained at baseline and within 3 weeks after axicabtagene ciloleucel (axi-cel) administration as indicated. Fresh frozen core biopsies at baseline were used technology(NanoString), using Clinical Research assays measure gene expression levels of multiple immune genes in a multiplex format. The assays were further developed to utilize minimal amounts of RNA from fresh frozen or formalin fixed tumor tissues. Predefined bioinformatics methods and cutoffs were applied to immune-mediated tumor regression genes to assess acquired immunity, including T cell cytotoxicity, T cell differentiation, T cell attraction, T cell adhesion, and immunosuppression, including immune targeting, angiogenesis inhibition, immune co-suppression, and cancer stem cells ( Figure 4; Galon J, et al. Immunity. 2013; 39: 11-26; www.haliodx.com / clinical-research-services / immunosignr / ). Bioinformatics methods include T cell-specific (effector T cells, Th1) genes, interferon pathway-related genes, chemokines, and immune checkpoints. High / Low The score cutoff of 21 was defined as the 25th percentile of scores observed in the sample. High scores indicate expression of immune-related genes that may be associated with tumor response.

[0232] Expression analysis and scoring were used to examine associations between TME features and response. A more extensive analysis was also performed using pre-specified gene sets, as shown in Table 2. The pre-specified 43 gene sets included 15, 21 and other genes, as well as all 763 genes in the PanCancer Immune Profiling Panel (nCounter Pan Cancer Immune Profiling Panel. https: / / www.nanostring.com / products / gene-expression-panels / hallmarks-cancer-gene-expression-panel-collection / pancancer-immune-profiling-panel). Fisher's exact test and Wilcoxon signed-rank test were used, and multiple testing was corrected by the false discovery rate (Benjamini-Hochberg).

[0233] Table 2. Predefined gene sets

[0234]

[0235]

[0236] Pre-existing immune features of the tumor microenvironment (TME) are associated with response to axicabtagene ciloleucel (axi-cel). Figure 5A As shown, the TME at baseline was evaluated in responders with a clinical follow-up of ≥9 months compared with non-responders. 21 score (P = 0.012). 85% (17 / 20) of the respondents had high 21 scores, while 80% (4 / 5) of non-responders had low 21 Ratings( Figure 5B and Figure 5C ). In a sensitivity analysis that included patients with delayed response at 12 months, The correlation between 21 and response had P = .053.

[0237] Compared with non-responders, the main immune-related genes upregulated at baseline in the TME of responders include CTLA4, CD3g, CD3e, CD27, SH2B2 and ICOSL. Compared with non-responders, other gene expressions in the TME are relatively reduced at baseline in responders, including MHC class II genes and cancer-testis antigens PRAME, MAGE, SSX. In view of the positive prognostic value of MHC class II expression in diffuse large B-cell lymphoma (Rimsza LM, et al., Blood. 2004; 103: 4251-4258), the correlation with MHC class II genes is surprising. Table 3 shows the differential expression of other genes from the expanded 763-gene pan-cancer immune spectrum analysis group in the TME in responders compared with non-responders.

[0238] Table 3 Differentially expressed genes in the TME of responders versus non-responders

[0239]

[0240]

[0241] Example 2: Baseline intratumoral T cell density as a prognostic of Axi-cel response

[0242] Additional post-hoc analyses of the Phase 2 ZUMA-1 study were designed to explore key pre-treatment features of the tumor immune microenvironment (TME), particularly CD3+ and CD8+ T cell density and and axi-cel response and immune gene expression (including 21).

[0243] Preconditioned tissue biopsy specimens were fixed in formalin and embedded in paraffin (FFPE) for analysis of CD3+ and CD8+ T cell densities (cells / mm 2 ).use Two consecutive FFPE sections (4 μm) were stained for CD3 and CD8 using the XT workstation, and the density of positive cell staining was determined using dedicated digital pathology software.

[0244] The analysis measures the density of CD8+ cytotoxic T cells and CD3+ T cells in resected or biopsied cancer samples and is performed on formalin-fixed paraffin-embedded tissue slides. The density (cells / mm) of T cell subsets (CD3+, CD8+) in tumor tissue biopsies before treatment (before chemotherapy conditioning) was analyzed by immunohistochemistry. 2 ).use CD3 and CD8 staining was performed on 2 consecutive FFPE sections (4 μm) using the XT workstation. Positive cell area was measured using dedicated digital pathology software. For each subject, CD3 and CD8 immunohistochemical staining of 25 baseline biopsy samples was scored and converted to CD3 using the HalonDx algorithm and analysis cutoff values ​​described by Galon et al. (J Pathol. 232: 199-209 (2014)). (a numerical index of T cell density). The observed median scores were defined as high and low The Welch's t test was used to compare the CR subjects with those with partial response, stable disease, and progressive disease. CD3 and CD8 levels.

[0245] Results: There was a higher density of intratumoral CD3+ and CD8+ T cells and high Most patients (all measured before treatment) were able to achieve CR ( FIG. 8A to FIG. 8C ). In general, higher was associated with achieving CR (P = 0.048; Fig. 8A and Figure 8B The intratumoral density of CD3+ and CD8+ T cells before treatment was positively correlated with the achievement of CR (P = 0.025 and 0.049, respectively; see Fig. 9A and Fig. 9B Patients who did not achieve CR mainly showed low density of CD3+ and CD8+ T cells in the tumor, as well as low ( Fig. 8A and Figure 8C ).

[0246] Evaluation of pre-treatment and 21, showing 82% agreement (95% CI, 65–93; r 2 =0.451; Fig.10 ), suggesting a potential relationship between a higher density of T cell infiltration and the permissive gene signature.

[0247] The findings of Examples 1 and 2 suggest that pre-treatment TME plays a key role in the response to CAR T cell therapy. Anti-CD19 CAR T therapy can overcome the 21 ratings or associated with poor prognosis.

[0248] Example 3: Tumor immune microenvironment

[0249] Further analysis of patient samples showed that CAR T cell expansion occurred within 2 weeks of treatment and was accompanied by an increase in cytokines in the immune program. Pharmacodynamic analysis showed that proliferation markers (IL-15 and IL-2), inflammatory markers (IL-6, CRP, SAA, IL-5, ferritin, IL-1Ra, IL-2Rα), immunomodulatory markers (GM-CSF, IFN-γ, IL-10), chemokines (IL-8, IP-10, MCP-1) and effector markers (granzyme B) all increased rapidly over baseline. Gene expression analysis showed that T cell-related genes (CD3ε, CD28 and CTLA4), innate immunity-related genes (MX1, ISG15 and MYD88) and B cell-related genes (CD19, CD79B, PAX5) before treatment may be associated with clinical outcomes ( Fig.11 ).

[0250] CAR T cells in the TME of Zuma-1 patients (7-21 days after treatment) were detected using in situ hybridization (ISH) for CAR T RNA and immunohistochemistry (IHC) using antibodies that recognize individual CAR epitopes as described in International Patent Application Publication Nos. WO2018 / 013563A1 and WO2018 / 053790A1. In patients with reduced tumor burden before treatment, an increase in sustained response rate at 1 year was observed. These data suggest that CAR T cell implantation corresponding to tumor burden can overcome large tumors. CAR T cell therapy is associated with changes in the TME. Transcript analysis of a pre-set 43 immune gene set showed upregulation in tumors 7-21 days after treatment. Increases in immune suppressive 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 were observed in response to treatment. Fig.12 Nanostring analysis showed elevated IL-15 and PD-L1 gene expression in subjects with complete or partial responses ( Fig.13A and Fig. 13B ).

[0251] Biopsy specimens from patients with large cell lymphoma and B-cell acute lymphoblastic leukemia (B-ALL) were evaluated for CD19 and PD-L1 expression. Among subjects who relapsed after Axi-cel, at least 1 / 3 of the subjects had tumors that were negative for CD19 expression. Analysis of B cells and immune-related molecules at progression identified relapses with CD19+ or CD19- tumor cells. At baseline, 94% (16 / 17) of evaluable patients were CD19+. Post-progression tumor biopsies from 21 evaluable patients showed that 33% were CD19- and 62% were PD-L1.

Claims

1. A method of treating a malignant tumor in a patient, 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 characteristics of the tumor microenvironment.

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

3. The method of claim 2, wherein the gene expression profiling comprises determining the expression levels of a specified set of genes.

4. The method of any of the preceding claims, comprising determining the expression level of one or more genes selected from the group consisting 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.

5. The method of any of the preceding claims, comprising determining the expression level of one or more genes selected from the group consisting of 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 the preceding claims, comprising determining the expression level of genes selected from the Pan Cancer Immune Profiling Panel.

7. The method of any one of the preceding claims, comprising determining the expression level of a B cell marker.

8. The method of any one of the preceding claims, comprising determining the expression level of a T cell marker.

9. The method of any one of the preceding claims, comprising determining the expression level of a specified group of genes, the specified group of genes comprising genes associated with an innate immune response.

10. The method of claim 9, wherein the specified set of genes includes markers for cytotoxic cells, dendritic cells, macrophages or granulocytes.

11. The method of any of the preceding claims, further comprising determining an immune score based on gene expression profiles and / or intratumoral T cell density.

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

13. The method of any of the preceding claims, wherein the effective dose comprises at least 1×10 6 CAR-positive live T cells per kg body weight.

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

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

16. The method of any of the preceding claims, wherein the malignancy is one or more of a solid tumor, a sarcoma, a carcinoma, a lymphoma, 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 myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), T cell lymphoma, B cell acute lymphoid leukemia ("BALL"), T cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid Dendritic cell neoplasms, 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, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasma cell neoplasms (e.g., plasma cell dyscrasias, 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 of the preceding claims, wherein the effective dose is optimized to increase the likelihood that the patient will respond to chimeric receptor therapy.

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

19. A method of determining whether a patient will respond to a 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; as well as (c) determining whether the patient will respond to chimeric receptor therapy based on the characteristics of the tumor microenvironment.

20. A method of treating a malignant tumor in a patient, comprising: (a) analyzing tumor biopsies from patients prior to chimeric receptor therapy to characterize the tumor microenvironment; (b) determining whether the patient will respond to chimeric receptor therapy based on characteristics of the tumor microenvironment; as well as (c) administering to the 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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