Creation of CAR modifiers for tumor treatment

Abstract

This disclosure provides modified immune cells or their precursors (e.g., T cells) comprising a first chimeric antigen receptor (CAR) capable of binding to human IL13Rα2, a second CAR capable of binding to EGFR or its isoform, and a dominant-negative TGFbβII receptor (DN-TGFbβRII). Compositions and therapeutic methods are also provided. TIFF2026509470000085.tif103170

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 119(e) of U.S. Patent Act to U.S. Provisional Patent Application No. 63 / 489,979, filed on 13 March 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Malignant gliomas, including grade IV gliomas, also known as glioblastoma (GBM), are the most common primary malignant brain tumors and are associated with high disease status and mortality. The invasive nature of the invasive proliferation of glioma cells in the central nervous system (CNS) makes complete resection impossible. Despite the best available treatments, including surgical resection, radiotherapy, chemotherapy, and alternating field tumor therapy systems, the median survival time is only 12–17 months for GBM patients and 2–5 years for grade III glioma patients.

[0003] Adoptive immunotherapy using redirected T cells is a viable strategy for treating these malignancies. Long disease-free survival was achieved in patients with refractory chronic lymphocytic leukemia after treatment with CD19-targeted chimeric antigen receptor-modified autologous T (CAR T) cells, and this strategy resulted in complete remission in 90% of patients with relapsed acute lymphoblastic leukemia (ALL). However, to date, the antitumor activity of CAR T cells in solid tumors has been far more modest. Humanized anti-EGFR variant III (EGFRvIII) CAR T cells (2173BBz) were previously used in a phase I clinical trial (NCT02209376) in 10 patients with relapsed GBM. There were clear changes in the tumor microenvironment after CAR T cell infusion, including a decrease in EGFRvIII target antigens associated with CAR T cell transport and in-situ functional activation. However, this study lacked the power to determine clinical response (median overall survival was 251 days). A recent report describes the use of repeated intratumoral and intrathecal injections of redirected T cells expressing the mutant IL13 cytokine IL13 zetakin, fused to the T cell signaling domain, in one patient with relapsed multifocal GBM, which resulted in complete tumor regression over 7.5 months.

[0004] Interleukin-13 receptor α2 (IL13Rα2) is expressed in different human tumor types but not in normal human tissues except for adult testes. IL13 signaling via IL13Rα2 plays a crucial role in cell migration and invasion. Previous studies have found that 82% of GBM cases express IL13Rα2. Neutralizing antibodies and drug-conjugated antibodies targeting IL13Rα2 inhibited tumor growth in xenograft mouse models. IL13Rα2-based tumor vaccines have also benefited pediatric glioma patients. IL13 zetakin-redirected T cells bound IL13Rα2 and induced limited clinical responses, but also bound IL13Rα1, which is expressed in some normal human tissues and showed adverse off-target effects.

[0005] Tumor heterogeneity and an immunosuppressive tumor microenvironment (TME) are major obstacles to chimeric antigen receptor (CAR) T-cell therapy in GBM. Site-specific declines in EGFRvIII after CAR T therapy are consistent with the observed intratumoral heterogeneity of this change in GBM. Importantly, immunohistochemical analysis of the tissue demonstrated the presence of an adaptive response within the GBM TME closely aligned with the time series of CAR T activation. IDO1, PD-L1, IL10, and TGFβ were all elevated in tumor tissue adjacent to CAR T cells after treatment. These immunomodulatory pathways play a crucial role in evading tumor immunity in many situations, suggesting that adaptive resistance develops within GBM, further blunting the immune response within the tumor.

[0006] In this field, there is a need for improved CAR T therapies that target multiple antigens and disrupt immunosuppressive signals within the TME. This invention addresses and satisfies this need. [Overview of the project]

[0007] As described herein, the present invention relates to modified immune cells or their precursors (e.g., T cells) comprising a first chimeric antigen receptor (CAR) capable of binding to human IL13Rα2, a second CAR capable of binding to EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Compositions and therapeutic methods are also provided.

[0008] In one aspect, the present invention is A first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain that binds human IL13Rα2, a transmembrane domain, and an intracellular domain, A second polynucleotide sequence encoding a second CAR, comprising a second antigen-binding domain that binds epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain, and The third polynucleotide sequence encoding the dominant-negative TGFβ type II receptor (DN-TGFβRII) It includes nucleic acids.

[0009] In one embodiment, the first and / or second antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, Fab, single-chain variable fragment (scFv), or a single-domain antibody.

[0010] In one embodiment, the first antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000002.tif4128 and HCDR3 contains the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and Includes.

[0011] In one embodiment, the first antigen-binding domain includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 8, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 9.

[0012] In one embodiment, the first antigen-binding domain is a single-stranded variable fragment (scFv) containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.

[0013] In one embodiment, the first polynucleotide sequence encodes a CAR that has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43.

[0014] In one embodiment, the second antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000003.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and Includes.

[0015] In one embodiment, the second antigen-binding domain includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32.

[0016] In one embodiment, the second antigen-binding domain is a single-stranded variable fragment (scFv) containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

[0017] In one embodiment, the second polynucleotide sequence encodes a CAR that has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75.

[0018] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0019] In one embodiment, the nucleic acid encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77 or 79.

[0020] In one embodiment, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and the transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0021] In one embodiment, the transmembrane domain includes the transmembrane domain of CD8.

[0022] In one embodiment, the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha.

[0023] In one embodiment, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain.

[0024] In one embodiment, the intracellular domain includes a co-stimulatory domain or variant thereof of a protein selected from the group consisting of proteins of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0025] In one embodiment, the intracellular domain includes a 4-1BB co-stimulatory domain.

[0026] In one embodiment, the intracellular signaling domain includes an intracellular domain selected from the group consisting of the cytoplasmic signaling domains of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0027] In one embodiment, the intracellular signaling domain includes the intracellular domain of CD3ζ.

[0028] In another aspect, the present invention is A nucleic acid comprising a first polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000004.tif4128 and HCDR3 includes the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and nucleic acids It includes.

[0029] In one embodiment, the antigen-binding domain includes a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

[0030] In one embodiment, the antigen-binding domain is an scFv containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.

[0031] In one embodiment, the CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43.

[0032] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0033] In another aspect, the present invention is A first polynucleotide sequence encoding a CAR, comprising an antigen-binding domain that binds to the epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain, and The second polynucleotide sequence encoding the dominant-negative TGFβ type II receptor (DN-TGFβRII) It includes nucleic acids.

[0034] In one embodiment, the antigen-binding domain includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32.

[0035] In one embodiment, the antigen-binding domain is an scFv containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

[0036] In one embodiment, the CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 35, 42, 43, or 75.

[0037] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0038] In one embodiment, the nucleic acid encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81, 83, or 85.

[0039] In another aspect, the present invention is A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first car, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000005.tif4128 and HCDR3 contains the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and It includes an antigen-binding domain, The second CAR, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000006.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and It includes an antigen-binding domain, The DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. It includes nucleic acids.

[0040] In another aspect, the present invention is A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The first car, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or 54, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Includes, The second CAR, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, It includes nucleic acids.

[0041] In another aspect, the present invention is A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The first CAR contains a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The second CAR contains a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70. It includes nucleic acids.

[0042] In another aspect, the present invention is A nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding IL13Rα2, a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The first polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO: 63, The second polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. It includes nucleic acids.

[0043] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0044] In one embodiment, a first polynucleotide sequence and a second polynucleotide sequence are separated by a linker.

[0045] In one embodiment, the second polynucleotide sequence and the third polynucleotide sequence are separated by a linker.

[0046] In one embodiment, the nucleic acid comprises a second polynucleotide sequence, a linker, and a first polynucleotide sequence, from 5' to 3'.

[0047] In one embodiment, the nucleic acid comprises a second polynucleotide sequence, a linker, a first polynucleotide sequence, a linker, and a third polynucleotide sequence, arranged from 5' to 3'.

[0048] In another aspect, the present invention encompasses vectors comprising nucleic acids as described in any of the above claims.

[0049] In one embodiment, the vector is an expression vector.

[0050] In one embodiment, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0051] In one embodiment, the vector of the above aspect or aspect or any aspect for the aspects disclosed herein further includes the EF-1a promoter.

[0052] In one embodiment, the vector in the above aspect or configuration or any aspect for the configuration disclosed herein further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0053] In one embodiment, the vector of the above-described aspect or aspect, or any aspect for any aspect disclosed herein, further includes a rev response element (RRE).

[0054] In one embodiment, the vector of the above-described aspect or aspect, or any aspect for any aspect disclosed herein, further comprises a cPPT sequence.

[0055] In one aspect, the vector is a self-inactivating vector.

[0056] In another aspect, the present invention includes modified immune cells or their progenitor cells comprising the nucleic acid described in any one of claims 1 to 38 or the vector described in any one of claims 39 to 46.

[0057] In another aspect, the present invention is A first chimeric antigen receptor (CAR) containing a first antigen-binding domain capable of binding IL13Rα2, A second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding epidermal growth factor receptor (EGFR) or its isoform, Dominant-negative TGFβ type II receptor (DN-TGFβRII) and This includes modified immune cells or their progenitor cells.

[0058] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first car, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains TIFF2026509470000007.tif4157 and HCDR3 has an amino acid sequence The heavy chain variable region, including TIFF2026509470000008.tif4128, A light chain variable region containing three light chain complementarity-determining regions (LCDRs), wherein LCDR1 is an amino acid sequence The light chain variable region includes TIFF2026509470000009.tif4128, where LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7) or QHHYSAPWT (SEQ ID NO: 18). Includes, The second CAR, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000010.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and including, This includes modified immune cells or their precursor cells.

[0059] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first car, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20. Includes, The second CAR, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. including, This includes modified immune cells or their precursor cells.

[0060] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR contains a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11. The second CAR contains a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71. This includes modified immune cells or their precursor cells.

[0061] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43, The second CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. This includes modified immune cells or their precursor cells.

[0062] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first car, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or 54, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Includes, The second CAR, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, This includes modified immune cells or their precursor cells.

[0063] In another aspect, the present invention is A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR contains an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The second CAR contains an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70. This includes modified immune cells or their precursor cells.

[0064] In another aspect, the present invention a modified immune cell or a progenitor cell thereof, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or an isoform thereof, and a dominant negative TGFβ type II receptor (DN-TGFβRII), where the first polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO: 63, where the second polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, comprising the modified immune cell or a progenitor cell thereof.

[0065] In certain embodiments, the DN-TGFβRII comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0066] In certain embodiments, the second CAR is wild-type EGFR (wtEGFR), mutant EGFR, EGFR [[ID=XX]] A289V [[ID=XX]]、EGFR[[ID=XX]] A289D [[ID=XX]]、EGFR[[ID=XX]] A289T [[ID=XX]]、EGFR[[ID=XX]] A289T [[ID=XX]]、EGFR[[ID=XX]] R108K [[ID=XX]]、EGFR[[ID=XX]] R108G [[ID=XX]]、EGFR[[ID=XX]] G598V [[ID=XX]]、EGFR[[ID=XX]] D126Y [[ID=XX]]、EGFR[[ID=XX]] C628F [[ID=XX]]、EGFR[[ID=XX]] R108K / A289V [[ID=XX]]、EGFR[[ID=XX]] R108K / D126Y [[ID=XX]]、EGFR[[ID=XX]] A289V / G598V [[ID=XX]]、EGFR[[ID=XX]] A289V / C628F Note: The tags A289V - A289V / C628F are repeated in the original text without clear indication of their meaning or intended use. They are left unchanged in the translation as per the instructions. If there is more context available for these tags, a more meaningful translation might be possible.EGFR isoforms selected from the group consisting of EGFR variant II, or any combination thereof, can be combined.

[0067] In one aspect, the modified cells are modified T cells.

[0068] In one aspect, the modified cells are autologous cells.

[0069] In one aspect, the modified cells are autologous cells derived from human subjects.

[0070] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0071] In another aspect, the present invention encompasses a pharmaceutical composition comprising a therapeutically effective amount of the modified cells described in any one of claims 47 to 61.

[0072] In another aspect, the present invention includes a method for treating a disease in a subject where such treatment is needed, comprising the step of administering an effective amount of the modified cells described in any one of claims 47 to 61 or the pharmaceutical composition described in claim 62 to the subject.

[0073] In one aspect, the disease is cancer.

[0074] In one respect, cancer is a glioma.

[0075] In one sense, cancer is an astrocytoma.

[0076] In one aspect, the cancer is a highly malignant astrocytoma.

[0077] In one respect, cancer is glioblastoma.

[0078] In another aspect, the present invention is A method for treating glioblastoma in subjects who require it, A first chimeric antigen receptor (CAR) containing a first antigen-binding domain capable of binding IL13Rα2, A second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding epidermal growth factor receptor (EGFR) or its isoform, Dominant-negative TGFβ type II receptor (DN-TGFβRII) and A method comprising the step of administering an effective amount of modified T cells containing the above. It includes.

[0079] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first car, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains TIFF2026509470000011.tif4157 and HCDR3 has an amino acid sequence Heavy chain variable region including TIFF2026509470000012.tif4128 Includes, The second CAR, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000013.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and including, It includes methods.

[0080] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first car, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20. Includes, The second CAR, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. including, It includes methods.

[0081] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first CAR contains an scFv that has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21 or SEQ ID NO: 22. The second CAR contains an scFv that has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71. It includes methods.

[0082] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 42 or SEQ ID NO: 43, The second CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. It includes methods.

[0083] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0084] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first car, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or 54, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Includes, The second CAR, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, It includes methods.

[0085] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first CAR contains an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The second CAR contains an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70. It includes methods.

[0086] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII) to the subject, The first CAR contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO: 63. The second CAR contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. It includes methods.

[0087] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0088] In another aspect, the present invention is A method for treating glioblastoma in a subject in need, comprising the step of administering to the subject an effective amount of modified T cells containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences to SEQ ID NO: 76 or 78. It includes. [Brief explanation of the drawing]

[0089] The above and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Figure 1] Schematic diagram of the dominant-negative TGFβ-RII CART-EGFR-IL13Rα2 construct. To inhibit the repressive signaling pathway, a cleaved TGF-β receptor II lacking the intracellular kinase domain at residue 199as was generated, preventing TGF-β receptor II from phosphorylating downstream signals. To overcome the dual challenges of antigen heterogeneity and the repressive tumor microenvironment, this was incorporated into a construct containing parallel CARs targeting EGFR and IL13Ra2. [Figure 2A]2A-2D: TGFβ1 is highly expressed in GBM. Figure 2A: TGFβ1 expression isolated by glioma histology based on RNA-seq data from the Cancer Genome Atlas (TCGA) database. The y-axis shows the relative expression level for each case. Figure 2B: Overall survival curves for high and low TGFβ1 expression groups from the Cancer Genome Atlas (TCGA) database, calculated by the upper and lower quartiles of TGFβ1 expression in Kaplan-Meier analysis. Figure 2C: TGFβ1 is expressed in GBM cell lines. Different glioblastoma cell lines were cultured for 3 days at a density of 3e6 cells per flask. Culture supernatants were collected to examine potential TGFβ secretion by glioblastoma cell lines. PC3 is one of the cell lines that has been shown to have high levels of secretion compared to GBM cell lines, and was used as a positive control. Figure 2D: Immunohistochemical staining with anti-TGF-β1 antibody on tissue sections derived from NSG mice intracranially transplanted with U87 and D270 gliomas. Spleen and cerebral cortex sections were used as positive and negative controls, respectively. Statistical significance was calculated by the Kruskal-Wallis test: **p<0.01, ***p<0.001, ****p<0.0001. [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 2C] Please refer to the explanation in Figure 2A. [Figure 2D] Please refer to the explanation in Figure 2A. [Figure 3A]Figures 3A-3D: T cells expressing the dnTGFβRII construct block immunosuppressive TGF-β signaling. Figure 3A: Schematic vector maps of the 806-Hu07-dnTGFβRII, 806-Hu07-mCherry, and dnTGFβRII-M5 CAR constructs. 806Hu07mCherry served as the corresponding control, and dnTGFβRII-M5 is a mesothelin CAR containing dnTGFBRII used as a suitable negative control. Figure 3B: Flow cytometry detection of T cell transduction. T cells were transduced with lentiviral vectors. CAR T cells had approximately equal expression among the three groups. TGF receptor II expression on CAR T cells was screened using an anti-TGFβ antibody. Expression was present in approximately 35% of CARs with dnTGFβRII. This confirmed that both CAR and dominant-negative TGFβRII were successfully expressed on T cells. The upper panel shows cells stained with biotinylated protein L for CAR expression, and the lower panel shows cells stained for TGFβRII. Figures 3C and D: TGF-β signaling induction in each group was evaluated via intracellular phosphorylated Smad2 / 3 and quantified in (D). dnTGFβRII blocks immunosuppressive TGFβ signaling at CAR-806-Hu07. [Figure 3B] Please refer to the explanation in Figure 3A. [Figure 3C] Please refer to the explanation in Figure 3A. [Figure 3D] Please refer to the explanation in Figure 3A. [Figure 4A] Figures 4A-4B: Short-term CAR cytotoxicity is unaffected by dnTGFβRII in vitro. Figure 4A: U87vIII-CBG luciferase target cells were co-cultured with effector T cells for 16 hours at different effector:target ratios using either 20 ng / ml hTGFβ1 or medium alone. Figure 4B: D270 target cells were co-cultured with effector T cells for 37 hours and screened by axion impedance. [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 5]806Hu07dnTGFβRII CAR T cells exhibit reduced PD1 expression in in vitro co-culture. Transduced T cells or UTD T cells (2 × 10⁵ cells per well in 100 μL of R10 medium) were co-cultured with target cells (2 × 10⁵ cells per well in 100 μL of R10 medium) in 96-well round-bottom tissue culture plates at 37°C and 5% CO₂ for 20 hours. T cells were collected, stained with CD3+PD1+, and analyzed using MFI. [Figure 6A] Figures 6A-6B: Human TGFβ1 does not reduce the activation of 806Hu07dnTGFβ CAR T cells in vitro. Transduced T cells or UTD T cells (2 × 10⁵ cells per well in 100 μL of R10 medium) were co-cultured with target cells (2 × 10⁵ cells per well in 100 μL of R10 medium) in 96-well round-bottom tissue culture plates at 37°C and 5% CO₂ for 16 hours. T cells were collected and stained with CAR+CD69+ or CAR+CD25+. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 7] dnTGFβRII enhanced CAR T cell proliferation with repeated stimulation. CAR T cells were restimulated weekly with the U87vIII tumor cell line using either culture supernatant or culture medium alone. Proliferation was compared between 806-Hu07-dnTGFβRII and 806-Hu07-mCherry CAR T cells in either culture supernatant or culture medium alone. [Figure 8] On days 7, 14, and 21, supernatants were collected from the growth co-culture plates and then stored at -80°C for future analysis of cytokine secretion. In addition to improved growth, 806-Hu07-dnTGFβRII CAR T cells also secreted greater amounts of effector cytokines, including IFN-γ, TNF-α, IL-12, GM-CSF, and IL-2, than 806-Hu07-mCherry CAR T cells. [Figure 9-1]Figure 9: 806-Hu07-dnTGFβRII cells transform into effector cell phenotypes. Figure 9A: In vitro long-term restorative assays were performed weekly to collect and stain T cells, and their phenotypic evolution was evaluated over time, specifically on days 0, 14, and 21. These changes were evaluated in different subsets: central memory (CM), naive (N), effector (E), and effector memory (EM) T cells. Figure 9B: The most significant changes occurred in the cohort exposed to 20 ng / ml TGF-β1. On day 14, 806-Hu07-dnTGFβRII cells showed a significantly more pronounced effector T cell phenotype than 806-Hu07-mCherry CAR T cells (p=0.0015). Figure 9C: By the final time point of day 21, 806-Hu07-dnTGFβRII cells showed an enhanced “effector memory” phenotype (p=0.0009). Figure 9D: CAR expression was compared in both 806-Hu07-dnTGFβRII and 806-Hu07-mCherry CAR T cells. These cells were subjected to either culture supernatant (+) or medium only (-), and this comparison was performed at the same time points as above. CAR expression was normalized to 41% at day 0 to initiate subsequent assays. [Figure 9-2] Please refer to the explanation in Figure 9-1. [Figure 10-1] Figures 10A-10D: The dnTGFβRII construct is safe in vivo. Figure 10A: Schematic diagram of the D270-CBG-GFP NSG mouse model (n=5 per cohort). 5e5 tumor cells were subcutaneously transplanted, and 7 days after tumor transplantation, each mouse was treated with intravenous infusion of CAR T cells. Tumor loading BLI was performed for 3-4 days. Figure 10B: Mouse body weight was measured every 3-4 days. Figure 10C: Tumor size was measured by caliper in length and width as tumor area. Figure 10D: Tumor regression was compared among each treated mouse using BLI. [Figure 10-2] Please refer to the explanation in Figure 10-1. [Figure 11-1]Figures 11A-11D: 806-Hu07-dnTGFβRII CAR T cells enhance the eradication of GBM tumors in vivo. Figure 11A: Schematic diagram of the U87vIII CBG-GFP NSG mouse model (n=5 per cohort). 5e5 tumor cells were transplanted intracranially, and 8 days after tumor transplantation, each mouse was treated with intravenous infusion of CAR T cells. Tumor loading BLI was performed for 3-4 days. Figure 11B: Survival curves for treated mice were calculated using a log-rank test adjusted for p-values ​​by Bonferroni correction for multiple comparisons, and survival was plotted using Kaplan-Meier curves based on time to the end of the experiment. Figures 11C-11D: Tumor size was compared among each treated mouse using BLI. [Figure 11-2] Please refer to the explanation in Figure 11-1. [Figure 11-3] Please refer to the explanation in Figure 11-1. [Modes for carrying out the invention]

[0090] Detailed explanation The present invention provides a composition and method comprising a modified immune cell or its precursor (e.g., a modified T cell) comprising a first chimeric antigen receptor (CAR) capable of binding to human IL13Rα2, a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβR). The provided composition and method are useful for treating cancer (e.g., glioma, high-grade astrocytoma, and glioblastoma).

[0091] Transforming growth factor β (TGFβ), a ligand for TGFβRII and a cytokine with a well-established role in tumor suppression, is highly expressed in GBM and increases after CART-EGFRvIII treatment. Many immunosuppressive features within the tumor microenvironment (TME), including macrophage M2 polarization, iTreg formation, and direct effects on the proliferation, differentiation, and function of naive and effector T cells, are attributed to TGFβ. Expression of cleaved type II TGFβ (TGFβRII), which lacks the kinase domain, acts as a dominant-negative TGFβ type II receptor (DN-TGFβR) to inhibit TGFβ signaling in cells. Expression of DN-TGFβR in human T cells with CAR via the use of lentiviral vectors enhanced the antitumor effect in vivo in a xenograft model of prostate cancer. Given the high TGFβ expression within GBM, and to mitigate TGFβ-mediated inhibitory activity, we combined a dominant-negative TGF-β receptor II (dnTGFbRII) with a dicistronic CART-EGFR-IL13Rα2 construct to generate a three-module construct, CART-EGFR-IL13Rα2-dnTGFbRII. While we do not wish to be constrained by theory, this approach was hypothesized to more effectively disrupt the resistance mechanisms observed in GBM. The data disclosed herein demonstrate that CART-EGFR-IL13Rα2-dnTGFbRII significantly increases T cell proliferation and enhances the functional response, particularly in TGFβ-rich tumor environments. Furthermore, in vivo studies confirmed the safety and efficacy of dnTGFβRII in collaboration with CAR in targeting GBM and eradicating GBM in an NSG mouse model.

[0092] Overcoming adaptive changes in the local tumor microenvironment and addressing antigen heterogeneity are necessary to improve the clinical efficacy of CAR T-targeted strategies. This disclosure demonstrates that a nicistronic CART construct works efficiently with cleaved TGFβ receptor II. There are several advantages to using dominant-negative TGFβRII in combination with CAR constructs containing CART-EGFR-IL13Rα2, including suppression of immunosuppressive TGFβ signaling and reduction of PD-1 expression by immune effector cells. In one embodiment, the DN-TGFβRII receptor enhances the proliferative capacity of CAR T cells in the context of chronic antigen stimulation without significant side effects, resulting in enhanced tumor eradication. In one embodiment, the three-modular CAR T construct disclosed herein, containing TGFβRII CART-EGFR-IL13Rα2, addresses the clinical challenges of antigen heterogeneity and immunosuppressive TME in GBM.

[0093] It should be understood that the methods described herein are not limited to the specific methods and experimental conditions disclosed herein, because such methods and conditions may change. It should also be understood that the terms used herein are intended solely to describe specific aspects and are not intended to limit them.

[0094] Furthermore, unless otherwise specified, the experiments described herein utilize conventional molecular biological, cell biological, and immunological techniques within the scope of the art of the art. Such techniques are well known to those skilled in the art and are adequately described in the literature. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008), including all appendices; and Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0095] A. definition Unless otherwise specified, scientific and technical terms used herein have meanings generally understood by those skilled in the art. In the event of potential ambiguity, the definitions provided herein shall prevail over dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of “or” shall mean “and / or” unless otherwise specified. The use of the term “including,” as well as other forms such as “includes” and “included,” is not limited to these.

[0096] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids described herein is well known and commonly used in the art. The methods and techniques provided herein are generally carried out, unless otherwise specified, according to conventional methods well known in the art, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as commonly achieved in the art or as described herein, according to the manufacturer's specifications. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and drug discovery chemistry described herein, as well as the experimental methods and techniques thereof, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

[0097] To facilitate easier understanding of this disclosure, the following terms are defined:

[0098] The article “one” is used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “element” means one or more elements.

[0099] As used herein, when referring to measurable values ​​such as quantity or duration, “about” is intended to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, such variations being suitable for carrying out the disclosed method.

[0100] As used herein, “activation” refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the induction of cytokine production and detectable effector function. The term “activated T cell” refers, among other things, to a T cell undergoing cell division.

[0101] As used herein, “alleviating” a disease means reducing the severity of one or more symptoms of the disease.

[0102] As used herein, the term “antigen” is defined as a molecule that elicits an immune response. This immune response may include antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen.

[0103] Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Therefore, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that induces an immune response may encode an "antigen" as used herein. Furthermore, those skilled in the art will understand that antigens do not need to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to induce a desired immune response. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by a "gene" at all. It is readily apparent that antigens may be synthesized and produced, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0104] As used herein, the term “self-derived” is intended to refer to any material that originates from an individual and is later reintroduced into the same individual.

[0105] A "costimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, including but not limited to proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0106] As used herein, “co-stimulatory signal” refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in upregulation or downregulation of T cell proliferation and / or important molecules.

[0107] "Disease" is a state of animal health in which the animal is unable to maintain homeostasis and, if the disease does not improve, the animal's health continues to deteriorate. In contrast, "disorder" in animals is a state of health in which the animal is able to maintain homeostasis, but the animal's health is less favorable than when the disorder is absent. If left untreated, a disorder does not necessarily lead to a further deterioration of the animal's health.

[0108] As used herein, the term “downregulation” refers to the reduction or elimination of gene expression of one or more genes.

[0109] "Effective dose" or "therapeutic effective dose" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological outcome or that provides a therapeutic or preventive benefit. Such outcomes may include, but are not limited to, an amount that, when administered to a mammal, elicits a detectable level of immunosuppression or tolerance compared to the immune response detected in the absence of the composition of the present invention. The immune response can be readily assessed by a number of methods known in the art. Those skilled in the art will understand that the amount of composition administered herein varies and can be readily determined based on many factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered.

[0110] "Code" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties that arise therefrom, which act as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, if the transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, that gene codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0111] As used herein, “endogenous” means any substance produced from or within an organism, cell, tissue, or system.

[0112] As used herein, the term “epitope” is defined as a small chemical molecule on an antigen that can trigger an immune response and induce a B-cell and / or T-cell response. An antigen may have one or more epitopes. Most antigens have many epitopes; i.e., they are polyvalent. Generally, epitopes are approximately about 10 amino acids and / or sugars in size. Preferably, epitopes are about 4 to 18 amino acids, more preferably about 5 to 16 amino acids, even more preferably 6 to 14 amino acids, more preferably about 7 to 12 amino acids, and most preferably about 8 to 10 amino acids. Those skilled in the art will understand that the overall three-dimensional structure, rather than a specific linear arrangement of molecules, is the primary criterion for antigen specificity and thus distinguishes one epitope from another. Based on this disclosure, the peptides used in the present invention may be epitopes.

[0113] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0114] As used herein, the term “expand” refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the number of T cells expanded ex vivo increases compared to the number initially present in the culture. In another embodiment, the number of T cells expanded ex vivo increases compared to other cell types in the culture. As used herein, the term “ex vivo” refers to cells taken from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0115] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence, driven by its promoter.

[0116] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) incorporating recombinant polynucleotides.

[0117] As used herein, “identity” refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. Two amino acid sequences are identical at the same position if, for example, the position in each of two polypeptide molecules is occupied by arginine. The identity or degree to which two amino acid sequences have the same residue at the same position in their alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences are identical (e.g., five positions in a polymer of 10 amino acids), the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0118] As used herein, the term “immune response” is defined as a cellular response to an antigen that occurs when lymphocytes recognize an antigen molecule as a foreign substance, induce antibody formation, and / or activate lymphocytes to eliminate the antigen.

[0119] The term "immunosuppressive" is used herein to refer to a reduction in the overall immune response.

[0120] "Isolated" means that it has been altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0121] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information to the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.

[0122] As used herein, the term “modified” means an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in many ways, including chemical, structural, and functional methods. Cells can be modified through the introduction of nucleic acids.

[0123] As used herein, the term “modulate” means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the subject in the absence of the treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. The term encompasses disrupting and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0124] In the context of this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0125] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it will be understood that this also includes RNA sequences where "U" replaces "T" (i.e., A, U, C, G).

[0126] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences, including degenerate versions of each other and those encoding the same amino acid sequence. Furthermore, the phrase "nucleotide sequences encoding a protein or RNA" may also include introns to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0127] Parenteral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intraventricular, intracranial, or intrasternal injection or infusion techniques. In one embodiment, the immunogenic compositions disclosed herein can be delivered to the CNS via intraventricular administration (e.g., by an Ommaya catheter).

[0128] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as by synthetic means.

[0129] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be contained in a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to one another by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, for example, and long chains, of which there are many types, commonly called proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0130] As used herein with respect to antibodies, the term “specifically binding” means an antibody that recognizes a particular antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to antigens of different allele types. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms “specific binding” or “specifically binding” may be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure of the chemical species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure rather than a broad protein. If an antibody is specific to epitope “A”, in a reaction involving label “A” and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0131] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating a signaling event, such as, but not limited to, signaling mediated by the TCR / CD3 complex. Stimulation can mediate changes in the expression of specific molecules, such as downregulation of TGF-beta and / or rearrangement of the cytoskeleton.

[0132] When used herein, "stimulating molecule" means a molecule on a T cell that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell.

[0133] As used herein, “stimulating ligand” means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), specifically binds to a congenital binding partner on a T cell (referred to herein as “stimulating molecule”), thereby mediating a primary response by T cells, including but not limited to activation, initiation of an immune response, and proliferation. Stimulating ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0134] The term “subject” is intended to include living organisms (e.g., mammals) from which an immune response can be induced. The “subject” or “patient” as used herein may be human or non-human mammals. Non-human mammals include, for example, livestock and pets, such as sheep, cattle, pigs, dogs, cats, and mouse mammals. Preferably, the subject is human.

[0135] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0136] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to the major histocompatibility complex molecule. The TCR is composed of an alpha (α) chain and a beta (β) chain heterodimer, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain consists of two extracellular domains, a variable domain, and a constant domain. In some embodiments, the TCR can be modified on any cell containing a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma-delta T cells.

[0137] As used herein, the term “therapeutic” means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of a disease state.

[0138] A "graft" refers to a biocompatible grid or donor tissue, organ, or cells to be transplanted. Examples of grafts include, but are not limited to, skin cells or tissues, bone marrow, and parenchymal organs such as the heart, pancreas, kidneys, lungs, and liver. A graft can also refer to any material to be administered to a host. For example, a graft can refer to nucleic acids or proteins.

[0139] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary target cells and their offspring.

[0140] As used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0141] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0142] Range: Throughout this disclosure, various aspects of the invention can be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose all the possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1-6 should be considered to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0143] B. Chimeric antigen receptor The present invention provides a chimeric antigen receptor (CAR) capable of binding to IL13Rα2 and / or epidermal growth factor receptor (EGFR) or an isoform thereof. The CAR of the present invention is used in combination with a dominant negative TGFβ type II receptor (DN-TGFβRII). In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to EGFR or an isoform thereof, a transmembrane domain, and an intracellular domain. Compositions and methods for modified immune cells or precursors thereof, such as modified T cells, comprising the CAR are also provided. Thus, in some embodiments, the immune cells are genetically modified to express the CAR. Nucleic acids encoding the CAR, vectors encoding the nucleic acids, and modified cells (e.g., modified T cells) comprising the CAR, vector, or nucleic acid are also provided.

[0144] The present CAR of the present invention comprises an antigen-binding domain capable of binding to IL13Rα2 and / or epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain. The present CAR of the present invention may optionally comprise a hinge domain. Thus, the present CAR of the present invention comprises an antigen-binding domain capable of binding to IL13Rα2 and / or epidermal growth factor receptor (EGFR), a hinge domain, a transmembrane domain, and an intracellular domain.

[0145] The antigen-binding domain can be functionally linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, described elsewhere herein for expression in cells. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is functionally linked to a second nucleic acid encoding the transmembrane domain and further functionally linked to a third nucleic acid sequence encoding the intracellular domain.

[0146] The antigen-binding domain described herein may be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR of the present invention. The CAR of the present invention may also include a hinge domain described herein. The CAR of the present invention may also include a spacer domain described herein. In some embodiments, each of the antigen-binding domain, transmembrane domain, and intracellular domain is separated by a linker.

[0147] In one embodiment, CAR can bind human IL13Rα2. In one embodiment, CAR can bind canine IL13Rα2. In one embodiment, CAR can bind both canine IL13Rα2 and human IL13Rα2.

[0148] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to human IL13Rα2, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence A heavy chain variable region containing TIFF2026509470000014.tif4128 and in which HCDR3 contains the amino acid sequence DHRDAMDY (SEQ ID NO: 4), and / or A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7). Includes.

[0149] In another embodiment, the CAR comprises an antigen-binding domain capable of binding IL13Rα2, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000015.tif4128 and HCDR3 contains the amino acid sequence QGTTALATRFFDV (SEQ ID NO: 15), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 contains the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence QHHYSAPWT (SEQ ID NO: 18), and Includes.

[0150] Acceptable variations in CAR sequences will be known to those skilled in the art. For example, in some embodiments, a CAR includes an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, or 18.

[0151] In one embodiment, a CAR capable of binding IL13Rα2 comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 8, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 9.

[0152] In one embodiment, a CAR capable of binding IL13Rα2 comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 19, and a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 20.

[0153] In one embodiment, a CAR capable of binding IL13Rα2 comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain includes an scFv having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, 11, 21, or 22.

[0154] In one embodiment, a CAR capable of binding IL13Rα2 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 42 or SEQ ID NO: 43 or SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO: 63 or SEQ ID NO: 75.

[0155] In one embodiment, CAR can bind to GBM stem cells.

[0156] In another embodiment, the CAR comprises an antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoforms, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000016.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and Includes.

[0157] In another embodiment, a CAR capable of binding EGFR or its isoforms comprises an antigen-binding domain comprising a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32.

[0158] In another embodiment, a CAR capable of binding EGFR or its isoforms includes an antigen-binding domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

[0159] In another aspect, a CAR capable of binding EGFR or an isoform thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75.

[0160] Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. In one aspect, the antigen-binding domain can bind IL13Rα2. In one aspect, the antigen-binding domain can bind human IL13Rα2. In one aspect, the antigen-binding domain can bind canine IL13Rα2. In one aspect, the antigen-binding domain can bind human IL13Rα2 and canine IL13Rα2. In one aspect, the antigen-binding domain can bind EGFR or an isoform thereof. In one aspect, the antigen-binding domain can bind wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V 、EGFR A289D 、EGFR A289T 、EGFR A289T 、EGFR R108K 、EGFR R108G 、EGFR G598V \(、EGFR\) D126Y 、EGFR C628F 、EGFR R108K / A289V 、EGFR R108K / D126Y 、EGFR A289V / G598V 、EGFR A289V / C628F 、and EGFR isoforms selected from the group consisting of EGFR variant II, or any combination thereof.

[0161] In one embodiment, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8. In another embodiment, the antigen-binding domain includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 9. In another embodiment, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19. In another embodiment, the antigen-binding domain includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 20.

[0162] In one aspect, the antigen-binding domain is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 1, HCDR2 contains the amino acid sequence of SEQ ID NO: 3, and HCDR3 contains the amino acid sequence of SEQ ID NO: 4, A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 5, LCDR2 contains the amino acid sequence of SEQ ID NO: 6, and LCDR3 contains the amino acid sequence of SEQ ID NO: 7. Includes.

[0163] In one aspect, the antigen-binding domain is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, and LCDR3 contains the amino acid sequence of SEQ ID NO: 18. Includes.

[0164] In one aspect, the antigen-binding domain is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 15, A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, and LCDR3 contains the amino acid sequence of SEQ ID NO: 18. Includes.

[0165] In one embodiment, the antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, Fab, a single-chain variable fragment (scFv), or a single-domain antibody. In one embodiment, the antigen-binding domain includes an scFv capable of binding IL13Rα2. In one embodiment, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 11. In one embodiment, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 22.

[0166] In one embodiment, the antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, Fab, a single-chain variable fragment (scFv), or a single-domain antibody. In one embodiment, the antigen-binding domain includes an scFv capable of binding to IL13Rα2.

[0167] Acceptable variations in antigen-binding domain sequences will be known to those skilled in the art. For example, in some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0168] Acceptable variations in antigen-binding domain sequences will be known to those skilled in the art. For example, in some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences described in SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0169] The antigen-binding domain may include, but is not limited to, any domain that binds to an antigen, and may include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. In some embodiments, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof. The choice of antigen-binding domain may depend on the type and number of antigens present on the surface of the target cell.

[0170] In some embodiments, the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single-stranded variable fragment (scFv). In some embodiments, the IL13Rα2-binding domain of the present invention is selected from the group consisting of an IL13Rα2-specific antibody, an IL13Rα2-specific Fab, and an IL13Rα2-specific scFv. In one embodiment, the IL13Rα2-binding domain is an IL13Rα2-specific antibody. In one embodiment, the IL13Rα2-binding domain is an IL13Rα2-specific Fab. In one embodiment, the IL13Rα2-binding domain is an IL13Rα2-specific scFv.

[0171] As used herein, the terms “single-stranded variable fragment” or “scFv” refer to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. The heavy chain (VH) and variable light chain (VL) are either directly linked or linked by a peptide-encoding linker that connects the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL. In some embodiments, the antigen-binding domain (e.g., IL13Rα2-binding domain) comprises an scFv having a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain comprises an scFv having a VL-linker-VH configuration from the N-terminus to the C-terminus. Those skilled in the art will be able to select a suitable configuration for use in the present invention.

[0172] Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. Linkers can ligate the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and International Publication No. 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. (GS) n (GSGGS) n(SEQ ID NO: 86), (GGGS) n (SEQ ID NO: 87), and (GGGGS) n Various linker sequences are known in the art, including but not limited to glycineserine (GS) linkers such as (SEQ ID NO: 88), where n is an integer of at least 1. Exemplary linker sequences are, but are not limited to, It may include amino acid sequences such as TIFF2026509470000017.tif24144. Those skilled in the art will be able to select a suitable linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), where VH and VL are amino acid sequences It is separated by a linker sequence containing TIFF2026509470000018.tif4128, which is a nucleic acid sequence It can be coded by TIFF2026509470000019.tif11142.

[0173] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing sequences encoding VH and VL, as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, and U.S. Patent Application Publications 20050196754 and 20050196754. The inhibitory antagonist scFv has been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 See 2(10:31-40). Stimulant agonists scFv are described (see, for example, Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0174] As used herein, "Fab" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and lacks an Fc region. For example, an antibody digested by the enzyme papain produces two Fab fragments and one Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind to the antigen).

[0175] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of the H chain and a light (L) chain linked by a disulfide bond for binding to the antigen, with the remaining H chain portions linked together. The "F(ab')2" fragment can be divided into two individual Fab' fragments.

[0176] In some embodiments, the antigen-binding domain may originate from the same species in which the CAR is ultimately used. For example, for use in humans, the antigen-binding domain of the CAR may contain a human antibody or a fragment thereof. In some embodiments, the antigen-binding domain may originate from a different species in which the CAR is ultimately used. For example, for use in humans, the antigen-binding domain of the CAR may contain a mouse antibody or a fragment thereof.

[0177] In some embodiments, the CARs of this disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on one target cell. In such embodiments, the CAR is a bispecific CAR or a multispecific CAR. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR containing one or more target-specific binding domains that have affinity for the same target antigen can bind different epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, the binding domains may be arranged in series and separated by a linker peptide. For example, in a CAR containing two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain via an oligo or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0178] transmembrane domain The CAR of the present invention may include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that can span the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is intended for insertion into a cell membrane, such as a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0179] In some embodiments, the transmembrane domain naturally associates with one or more domains in the CAR. In some embodiments, the transmembrane domain may be selected to avoid binding such domain to the transmembrane domains of identical or different surface membrane proteins, or may be modified by one or more amino acid substitutions, in order to minimize interaction with other members of the receptor complex.

[0180] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-binding protein or transmembrane protein, such as a type I transmembrane protein. If the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates CAR insertion into the cell membrane, such as an artificial hydrophobic sequence. Examples of transmembrane domains particularly useful in the present invention include, but are not limited to, transmembrane domains derived from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 (i.e., including at least their transmembrane regions), or transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). In one embodiment, the transmembrane domain includes the transmembrane domain of CD8. In one embodiment, the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha.

[0181] In some embodiments, the transmembrane domain may be synthetic and in which case it mainly comprises hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0182] The transmembrane domains described herein may be combined with any of the antigen-binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in this CAR.

[0183] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates appropriate protein folding for the CAR. The hinge region is an optional component of the CAR. The hinge region may include a domain selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. Examples of hinge regions, but not limited to, include the CD8a hinge, an artificial hinge made from a polypeptide which may be as small as three glycine (Gly) molecules, and the CH1 and CH3 domains of IgG (such as human IgG4).

[0184] In some embodiments, the CAR of this disclosure includes a hinge region connecting an antigen-binding domain to a transmembrane domain, to which the transmembrane domain then connects to an intracellular domain. The hinge region can preferably support the antigen-binding domain in recognizing and binding to a target antigen on a target cell (see, e., Hudecek et al., Cancer Immunol. Res. (2015) 3(2):125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell such as a tumor cell (Hudecek et al., ibid.). The flexibility of the hinge region allows the hinge region to assume many different three-dimensional structures.

[0185] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0186] The hinge region can have a length of approximately 4 to 50 amino acids, for example, approximately 4aa to 10aa, approximately 10aa to 15aa, approximately 15aa to 20aa, approximately 20aa to 25aa, approximately 25aa to 30aa, approximately 30aa to 40aa, or approximately 40aa to 50aa. In some embodiments, the hinge region can have a length greater than 5aa, greater than 10aa, greater than 15aa, greater than 20aa, greater than 25aa, greater than 30aa, greater than 35aa, greater than 40aa, greater than 45aa, greater than 50aa, greater than 55aa, or more.

[0187] A suitable hinge region can be easily selected and can be any of a number of suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, for example, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. A suitable hinge region can have a length greater than 20 amino acids (e.g., 30, 40, 50, 60, or more amino acids).

[0188] For example, the hinge region is made of glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n (GSGGS) n (SEQ ID NO: 86) and (GGGS) n (SEQ ID NO: 87) includes glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than alanine and is far less restrictive than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:73-142). An exemplary hinge region is, It may include, but is not limited to, amino acid sequences such as TIFF2026509470000020.tif17145.

[0189] In some embodiments, the hinge region is the immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are publicly known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4):1779-1789. As a non-limiting example, the immunoglobulin hinge region may have the following amino acid sequence: TIFF2026509470000021.tif4148 (see, for example, Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); It can include one of the following: TIFF2026509470000022.tif31138, etc.

[0190] The hinge region may contain the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the hinge region may contain one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may have a hinge region sequence The Tyr can be substituted to include EPKSCDKTYTCPPCP (SEQ ID NO: 104), see, for example, Yan et al., J. Biol. Chem. (2012) 287:5891-5897. In one embodiment, the hinge region may contain an amino acid sequence derived from human CD8, or a variant thereof.

[0191] intracellular domain The CAR of the present invention also includes an intracellular domain. In one embodiment, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. The intracellular domain of the CAR is responsible for activating at least one effector function of the cell in which the CAR is expressed (e.g., an immune cell). The intracellular domain transmits effector function signals, instructing the cell (e.g., an immune cell) to perform its specific function, such as damaging and / or destroying target cells.

[0192] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of surface receptors, costimulatory molecules, and any molecules that act in coordination to initiate signal transduction in T cells, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0193] Examples of intracellular domains include, but are not limited to, the ζ chain or any of its homologs of the T cell receptor complex, such as the η chain, FcsRIγ and β chains, MB 1(Iga) chain, B29(Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be the human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof.

[0194] In one embodiment, the intracellular domain of CAR includes any portion of one or more co-stimulatory molecules, such as CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional ability, and at least one signaling domain from any combination thereof. The intracellular domain includes a co-stimulatory domain or variant thereof of a protein selected from the group consisting of proteins of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). In one embodiment, the intracellular domain includes a 4-1BB co-stimulatory domain.

[0195] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon RIb), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Examples include, but are not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequences of costimulatory molecules having the same functional capacity, and any combination thereof, one or more molecules or receptor-derived fragments or domains.

[0196] Further examples of intracellular domains include, but are not limited to, the intracellular signaling domains of various other immune signaling receptors of several types, including, but not limited to, first, second, and third-generation T cell signaling proteins, including CD3, B7 family costimulatory molecules, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Furthermore, intracellular signaling domains may include signaling domains used by NK and NKT cells, such as NKp30(B7-H6) (see, e.g., Zhang et al., J.Immunol.(2012)189(5):2290-2299), DAP12 (see, e.g., Topfer et al., J.Immunol.(2015)194(7):3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z signaling domains (see, e.g., Hermanson and Kaufman, Front.Immunol.(2015)6:195).

[0197] In one embodiment, the intracellular domain includes an intracellular signaling domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In one embodiment, the intracellular domain includes the intracellular domain of CD3ζ.

[0198] The intracellular domain suitable for use in the present invention of this CAR includes any desired signaling domain that provides a clear and detectable signal in response to activation of the CAR (i.e., activated by an antigen and a dimerizing agent) (e.g., increased production of one or more cytokines by cells; altered transcription of target genes; altered protein activity; altered cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of cellular signaling responses, etc.). In some embodiments, the intracellular domain includes at least one ITAM motif (e.g., 1, 2, 3, 4, 5, 6, etc.) as described below. In some embodiments, the intracellular domain includes a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular domain is not covalently bound to the membrane-bound CAR but is instead diffused into the cytoplasm.

[0199] The intracellular domain suitable for use in the present invention's CAR contains an intracellular signaling polypeptide containing an immune receptor tyrosine activation motif (ITAM). In some embodiments, the ITAM motif is repeated twice in the intracellular domain, and the presence of the first and second ITAM motifs is separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular domain of the present CAR contains three ITAM motifs.

[0200] In some embodiments, the intracellular domain includes a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine activation motif (ITAM) such as, but not limited to, Fc-gamma-RI, Fc-gamma-RIIA, Fc-gamma-RIIC, Fc-gamma-RIIIIA, and FcRL5 (see, for example, Gillis et al., Front.Immunol. (2014) 5:254).

[0201] A suitable intracellular domain may be an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide. For example, a suitable intracellular domain may be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Therefore, a suitable intracellular domain does not need to contain the entire sequence of the protein from which it originates. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0202] In one embodiment, the intracellular domain is derived from DAP12 (TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR-related protein; TYRO protein tyrosine kinase-binding protein; also known as killer-activating receptor associated protein; killer-activating receptor-associated protein, etc.). In one embodiment, the intracellular domain is derived from FCER1G (FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc epsilon RI gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; also known as high affinity gamma chain of immunoglobulin E receptor, etc.). In one embodiment, the intracellular domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one embodiment, the intracellular domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one embodiment, the intracellular domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).In one embodiment, the intracellular domain is derived from CD79A (also known as the B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.). In one embodiment, the intracellular domain suitable for use in the FN3 CAR of this disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular domain suitable for use in the FN3 CAR of this disclosure comprises a ZAP70 polypeptide. In several embodiments, the intracellular domain comprises a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular domain in the CAR comprises a cytoplasmic signaling domain of human CD3 zeta.

[0203] Typically, the entire intracellular domain can be used, but often it is not necessary to use the entire chain. To the extent that a cleaved portion of the intracellular domain is used, such a cleaved portion can be used in place of the intact chain, as long as it transmits the effector functional signal. The intracellular domain contains any cleaved portion of the intracellular domain sufficient to transmit the effector functional signal.

[0204] The intracellular domains described herein can be combined with any of the antigen-binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in CAR.

[0205] (Table 1) Sequence used in the present invention TIFF2026509470000023.tif178145TIFF2026509470000024.tif224145TIFF2026509470000025.tif223145TIFF2026509470000026.tif218145TIFF2026509470000027.tif223145TIFF2026509470000028.tif223145TIFF2026509470000029.tif226145TIFF2026509470000030.tif223145TIFF2026509470000031.tif226145TIFF2026509470000032.tif222145TIFF2026509470000033.tif226145TIFF2026509470000034.tif226145TIFF2026509470000035.tif226145TIFF2026509470000036.tif226145TIFF2026509470000037.tif226145TIFF2026509470000038.tif226145TIFF2026509470000039.tif226145TIFF2026509470000040.tif226145TIFF2026509470000041.tif42145

[0206] C. Series and parallel dual-singularity CAR Serial CARs, cells containing serial CARs (e.g., T cells), amino acid sequences containing serial CARs, and nucleic acids encoding serial CARs are also provided herein. A serial CAR comprises two antigen-binding domains separated by a linker, the linker being linked to a transmembrane domain and an intracellular domain (e.g., 4-1BB and / or CD3ζ). In one aspect, a serial CAR comprises a first antigen-binding domain (e.g., a first scFv) separated by a linker from a second antigen-binding domain (e.g., a second scFv), followed by a transmembrane domain and an intracellular domain (e.g., 4-1BB and / or CD3ζ). The first and second antigen-binding domains can bind two different antigens. For example, an exemplary serial CAR comprises a first antigen-binding domain containing an scFv capable of binding IL13Rα2, and a second antigen-binding domain containing an scFv capable of binding EGFR.

[0207] The linker in the serial CAR linking the first and second antigen-binding domains can be of various sizes, e.g., any number of amino acid lengths. For example, the linker can be 1 amino acid length, 2 amino acid lengths, 3 amino acid lengths, 4 amino acid lengths, 5 amino acid lengths, 6 amino acid lengths, 7 amino acid lengths, 8 amino acid lengths, 9 amino acid lengths, 10 amino acid lengths, 11 amino acid lengths, 12 amino acid lengths, 13 amino acid lengths, 14 amino acid lengths, 15 amino acid lengths, 16 amino acid lengths, 17 amino acid lengths, 18 amino acid lengths, 19 amino acid lengths, 20 amino acid lengths, 21 amino acid lengths, 22 amino acid lengths, 23 amino acid lengths, 24 amino acid lengths, or 25 amino acid lengths. In one embodiment, the serial CAR includes a linker that is 5 amino acid lengths long. In one embodiment, the serial CAR includes the amino acid sequence of SEQ ID NO: 77 and may be encoded by the nucleotide sequence of SEQ ID NO: 76. In one embodiment, the serial CAR includes a linker that is 10 amino acid lengths long. In one embodiment, the serial CAR may include an amino acid sequence with SEQ ID NO: 79 and be encoded by a nucleotide sequence with SEQ ID NO: 78. In one embodiment, the serial CAR may include a linker that is 15 amino acids long. In one embodiment, the serial CAR may include an amino acid sequence with SEQ ID NO: 81 and be encoded by a nucleotide sequence with SEQ ID NO: 80.

[0208] Parallel CARs, cells containing parallel CARs (e.g., T cells), amino acid sequences containing parallel CARs, and nucleic acids encoding parallel CARs are also provided herein. A parallel CAR comprises two distinct CARs linked by a cleavable linker (e.g., a 2A linker). For example, an exemplary parallel CAR comprises a first antigen-binding domain (e.g., scFv) linked to a first transmembrane domain and a first intracellular domain, and a second antigen-binding domain (e.g., scFv) linked to a second transmembrane domain and a second intracellular domain, along with a cleavable linker (e.g., a 2A linker). When the nucleic acid is expressed in a cell, the linker (e.g., a 2A linker) is cleaved, and two distinct CARs are expressed on the cell surface. In one embodiment, the parallel CAR comprises a first CAR capable of binding IL13Rα2 and a second CAR capable of binding EGFR.

[0209] D. Nucleic acids and expression vectors This disclosure provides nucleic acids encoding a first CAR capable of binding IL13Rα2a, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβII receptor (DN-TGFβRII). The nucleic acids of this disclosure may comprise polynucleotide sequences encoding any one or more of the CARs disclosed herein.

[0210] In one embodiment, the nucleic acid of the present disclosure comprises a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain for binding human IL13Rα2, a transmembrane domain, and an intracellular domain; a second polynucleotide sequence encoding a second CAR comprising a second antigen-binding domain for binding epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain; and a third polynucleotide sequence encoding a dominant-negative TGFβII receptor (DN-TGFβRII).

[0211] In one embodiment, the nucleic acid of the Disclosure comprises a first polynucleotide sequence encoding a CAR capable of binding to IL13Rα2 and a second polynucleotide sequence encoding a dominant-negative TGFβII receptor (DN-TGFβRII). In one embodiment, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence A heavy chain variable region containing TIFF2026509470000042.tif4128 and in which HCDR3 contains the amino acid sequence DHRDAMDY (SEQ ID NO: 4), and / or A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7). Includes.

[0212] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβII receptor (DN-TGFβRII), and the antigen-binding domain includes a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, and / or a light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48.

[0213] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβ-II receptor (DN-TGFβRII), and the antigen-binding domain of the CAR is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64 or 69.

[0214] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) and a dominant-negative TGFβ type II receptor (DN-TGFβRII) capable of binding IL13Rα2, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000043.tif4128 and HCDR3 contains the amino acid sequence QGTTALATRFFDV (SEQ ID NO: 15), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 contains the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence QHHYSAPWT (SEQ ID NO: 18), and nucleic acids It is also offered.

[0215] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβII receptor (DN-TGFβRII), and the antigen-binding domain of the CAR includes a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54, and / or a light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.

[0216] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβ-II receptor (DN-TGFβRII), and the antigen-binding domain of the CAR is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 or 66.

[0217] In another context, this disclosure is: A nucleic acid comprising a polynucleotide sequence encoding a CAR capable of binding to IL13Rα2 and a dominant-negative TGFβII receptor (DN-TGFβRII), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48. To provide.

[0218] In another context, this disclosure is: A nucleic acid comprising a polynucleotide sequence encoding a CAR capable of binding to IL13Rα2 and a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54, and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. To provide.

[0219] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 or SEQ ID NO: 66, or SEQ ID NO: 62 or SEQ ID NO: 63, and a second polynucleotide sequence encoding DN-TGFβRII.

[0220] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0221] Also provided are nucleic acids comprising a first polynucleotide sequence encoding a CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a second polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII).

[0222] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβII receptor (DN-TGFβRII), wherein the antigen-binding domain of the CAR includes a heavy chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and / or a light chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32.

[0223] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβ-II receptor (DN-TGFβRII), and the antigen-binding domain is an scFv containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

[0224] In one embodiment, the nucleic acid encodes a CAR and a dominant-negative TGFβII receptor (DN-TGFβRII), and the CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75.

[0225] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0226] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first and second CARs each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0227] In one embodiment, the antigen-binding domain of the first CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000044.tif4128 and HCDR3 contains the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and Includes.

[0228] In one embodiment, the antigen-binding domain of the first CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000045.tif4128 and HCDR3 contains the amino acid sequence QGTTALATRFFDV (SEQ ID NO: 15), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 contains the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence QHHYSAPWT (SEQ ID NO: 18), and Includes.

[0229] In one embodiment, the antigen-binding domain of the first CAR includes a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, and / or a light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48.

[0230] In one embodiment, the antigen-binding domain of the first CAR includes a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54, and / or a light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.

[0231] In one embodiment, the antigen-binding domain of the first CAR is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 138, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66.

[0232] In one embodiment, the first polynucleotide sequence includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO: 63.

[0233] In one embodiment, the antigen-binding domain of the second CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000046.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGINLDD (SEQ ID NO: 143) or HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and Includes.

[0234] In one embodiment, the antigen-binding domain of the second CAR includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32. In one embodiment, the antigen-binding domain of the second CAR includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 8, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 9. In one embodiment, the antigen-binding domain of the second CAR includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 19, and / or a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 20.

[0235] In one embodiment, the antigen-binding domain of the second CAR is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70 or SEQ ID NO: 71. In another embodiment, the antigen-binding domain of the second CAR is a single-stranded variable fragment (scFv) containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 or SEQ ID NO: 66 or SEQ ID NO: 64.

[0236] In one embodiment, the second polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In one embodiment, the second polynucleotide sequence encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or SEQ ID NO: 75.

[0237] A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains TIFF2026509470000047.tif4156 and HCDR3 has an amino acid sequence The heavy chain variable region, including TIFF2026509470000048.tif4128, A light chain variable region containing three light chain complementarity-determining regions (LCDRs), wherein LCDR1 is an amino acid sequence The light chain variable region includes TIFF2026509470000049.tif4128, where LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7) or QHHYSAPWT (SEQ ID NO: 18). Including the second CAR, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000050.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGINLDD (SEQ ID NO: 143) or HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and nucleic acids It is also offered.

[0238] A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first CAR comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or 54, and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48 or 58, and the second CAR comprises SEQ ID NO: Nucleic acid comprising a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74. It is also offered.

[0239] A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first CAR comprises a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 69, 64, 65, or 66, and the second CAR comprises a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70. It is also offered.

[0240] A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or 53 or 62 or 63, and the second polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. It is also offered.

[0241] In some embodiments, the nucleic acids of the Disclosure are provided for the production of CARs as described herein, for example, in mammalian cells. In some embodiments, the nucleic acids of the Disclosure provide amplification of nucleic acids encoding CARs.

[0242] In some embodiments, the nucleic acids of the Disclosure comprise a first polynucleotide sequence and a second polynucleotide sequence. In some embodiments, the nucleic acids of the Disclosure comprise a first polynucleotide sequence, a second polynucleotide sequence and a third polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker, and / or the second and third polynucleotide sequences may be separated by a linker. The linker for use in the Disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or dicistronic sequence), and they are translated as polyproteins that dissociate into distinct protein components. For example, a linker for use in the nucleic acids of the Disclosure comprising an IL13Rα2CAR coding sequence and an EGFR CAR coding sequence allows IL13Rα2CAR and EGFR CAR to be translated as polyproteins that dissociate into separate CARs. In some embodiments, the nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In one embodiment, the nucleic acid comprises a second polynucleotide sequence, a linker, and a first polynucleotide sequence from 5' to 3'. In another embodiment, the nucleic acid comprises a first polynucleotide sequence, a linker, a second polynucleotide sequence, a linker, and a third polynucleotide sequence from 5' to 3'. In yet another embodiment, the nucleic acid comprises a second polynucleotide sequence, a linker, a first polynucleotide sequence, a linker, and a third polynucleotide sequence from 5' to 3'.

[0243] In some embodiments, the linker comprises a nucleic acid sequence encoding an intrasequence ribosome entry site (IRES). As used herein, “intrasequence ribosome entry site” or “IRES” refers to an element that facilitates direct intrasequence ribosome entry into a start codon, such as ATG, in a protein-coding region, thereby resulting in cap-independent translation of the gene. IRESs can be obtained from viral or cellular mRNA sources, e.g., immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and a variety of intrasequence ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs that can be obtained from, for example, cardioviruses, rhinoviruses, aftviruses, HCV, Friend mouse leukemia virus (FrMLV) and Moloney mouse leukemia virus (MoMLV). Those skilled in the art will be able to select an appropriate IRES for use in the present invention.

[0244] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that enables multiple proteins to be encoded as a polyprotein, which dissociates into component proteins during translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae family of viridae, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asigna virus (TaV), and porcine rhinitis virus-1 (PTV-1); as well as carioviruses such as tylovirus and encephalomyocarditis virus. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those skilled in the art will be able to select an appropriate self-cleaving peptide for use in the present invention.

[0245] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease present in the trans-Golgi and processes protein precursors before their secretion. Furin cleaves at the COOH terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including but not limited to Arg-X1-Lys-Arg or Arg-X1-Arg-Arg, X2-Arg-X1-X3-Arg (SEQ ID NO: 108) and Arg-X1-X1-Arg, e.g., Arg-Gln-Lys-Arg (SEQ ID NO: 109), where X1 is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0246] In some embodiments, the linker includes a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, linkers containing nucleic acid sequences encoding furin and F2A, linkers containing nucleic acid sequences encoding furin and E2A, linkers containing nucleic acid sequences encoding furin and P2A, and linkers containing nucleic acid sequences encoding furin and T2A. Those skilled in the art will be able to select a suitable combination for use in the present invention. In such embodiments, the linker may further include a spacer sequence between furin and the 2A peptide. Various spacer sequences are known in the art, including but not limited to glycineserine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO: 86), and (GGGS)n (SEQ ID NO: 87), where n is an integer of at least 1. Exemplary spacer sequences are, but are not limited to, This may include amino acid sequences such as TIFF2026509470000051.tif11128. Those skilled in the art will be able to select a suitable spacer sequence for use in the present invention.

[0247] In some embodiments, the nucleic acids of this disclosure can be functionally ligated to transcriptional regulatory elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those skilled in the art.

[0248] In one embodiment, a nucleic acid encoding an exogenous CAR is functionally linked to the promoter. In another embodiment, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0249] Regarding expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambdaP, and trc. Regarding expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus pre-early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeat sequences of retrovirus origin; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated from and derived from many organisms, eukaryotes and prokaryotes. Modifications of reversible promoters derived from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) are well known in the art.Such reversible promoters, and systems that include further regulatory proteins based on such reversible promoters, include, but are not limited to, alcohol-regulating promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulating promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulating promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulating promoters (e.g., metallothionein promoter system, etc.), pathogenicity-related regulatory promoters (e.g., salicylic acid-regulating promoter, ethylene-regulating promoter, benzothiadiazole-regulating promoter, etc.), temperature-regulating promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulating promoters, synthesis-inducible promoters, etc.).

[0250] In some embodiments, the promoter may be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc.Natl.Acad.Sci.USA(1993)90:7739; and Marodon et al.(2003)Blood 101:3416. Another example is the use of a CD8 gene promoter. NK cell-specific expression can be achieved by using the NcrI(p46) promoter; see, e.g., Eckelhart et al.Blood(2011)117:1565.

[0251] For expression in yeast cells, suitable promoters include constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter; or moduloable promoters, such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (for example, for use in the genus Pichia). The selection of appropriate vectors and promoters is well within the realm of those skilled in the art. Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; trp promoter; lac operon promoter; hybrid promoters, e.g., lac / tac hybrid promoter, tac / trc hybrid promoter, trp / lac promoter, T7 / lac promoter; trc promoter; tac promoter, etc.; araBAD promoter; ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637); pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1):86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21):10079-83); nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813); and other in vivo regulatory promoters (e.g., Dunstan). et al., Infect.Immun.(1999)67:5133-5141; McKelvie et al., Vaccine(2004)22:3243-3255; and Chatfield et al., Biotechnol.See (1992)10:888-892); sigma70 promoter, e.g., consensus sigma70 promoter (e.g., see GenBank accession numbers AX798980, AX798961 and AX798183); stationary phase promoter, e.g., dps promoter, spv promoter, etc.; promoters derived from pathogenic island SPI-2 (e.g., see International Publication No. 96 / 17951); actA promoter (e.g., see Shetron-Rama et al., Infect.Immun.(2002)70:1087-1096); rpsM promoter (e.g., see Valdivia and Falkow Mol.Microbiol.(1996)22:367); tet promoter (e.g., Hillen, W. and Wissmann, A.(1989)In Saenger, W. and Heinemann, U.(eds), Topics in Molecular and See Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol.10, pp.143-162); SP6 promoter (e.g., Melton et al., Nucl. Acids Res.)Examples of operators suitable for use in prokaryotes such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7, and P-lambda. Non-exclusive examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (e.g., deBoer et al., Proc.Natl.Acad.Sci.USA(1983)80:21-25).

[0252] Other examples of suitable promoters include the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence functionally linked to it. Other constitutive promoter sequences may also be used, for example, but are not limited to, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) or human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus pre-early promoter, the Roussarcoma virus promoter, the EF-1 alpha promoter, and human gene promoters, for example, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a functionally linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is undesirable. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. In one embodiment, the present invention provides a polynucleotide sequence encoding a CAR (e.g., bispecific CAR, serial CAR, parallel CAR, etc.) that includes an inductive promoter. In one embodiment, the inductive promoter promotes the expression of a functionally linked sequence (e.g., a CAR) after T cell activation. T cells (e.g., CAR T cells) can be modified with this promoter to express a designed RNA or amino acid.

[0253] In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched by induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art, and for example, induction of an irreversible switch may utilize Cre-lox-mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc.Natl.Acad.Sci.USA(2000)28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art may be used to construct a promoter that can be irreversibly switched. The methods, mechanisms, and requirements for site-directed recombination described elsewhere in this specification are used in the production of irreversibly switched promoters and are well known in the art; see, for example, Grindley et al. Annual Review of Biochemistry (2006) 567-605, and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosure of which is incorporated herein by reference.

[0254] In some embodiments, the nucleic acids of the Disclosure further comprise a nucleic acid sequence encoding a CAR-inducible expression cassette. In one embodiment, the CAR-inducible expression cassette is for the production of a transgenic polypeptide product released in response to CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acids of the Disclosure further comprise a nucleic acid sequence encoding a cytokine functionally linked to a T cell activation-responsive promoter. In some embodiments, the cytokine functionally linked to the T cell activation-responsive promoter resides on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0255] The nucleic acids of this disclosure may be present in expression vectors and / or cloning vectors. Expression vectors may include selection markers, origins of replication, and other features that provide vector replication and / or maintenance. Suitable expression vectors include, for example, plasmids and viral vectors. Numerous suitable vectors and promoters are known to those skilled in the art; many are commercially available for constructing recombinant constructs of interest. The following vectors are provided as examples and should not be construed as limiting: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0256] Expression vectors generally have a convenient restriction site located near the promoter sequence to provide insertion of a nucleic acid sequence encoding a heterologous protein. Functional selection markers may be present in the expression host. Appropriate expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (e.g., Li et al., Invest.Opthalmol.Vis.Sci.(1994)35:2543-2549; Borras et al., Gene Ther.(1999)6:515-524; Li and Davidson, Proc.Natl.Acad.Sci.USA(1995)92:7700-7704; Sakamoto et al., H.Gene Ther.(1999)5:1088-1097; International Publication No. 94 / 12649, International Publication No. 93 / 03769; International Publication No. 93 / 19191; International Publication No. 94 / 28938); See International Publication Nos. 95 / 11984 and International Publication Nos. 95 / 00655; Adeno-associated viruses (e.g., Ali et al., Hum.Gene Ther.(1998)9:81-86, Flannery et al., Proc.Natl.Acad.Sci.USA(1997)94:6916-6921; Bennett et al., Invest.Opthalmol.Vis.Sci.(1997)38:2857-2863; Jomary et al., Gene Ther.(1997)4:683-690, Rolling et al., Hum.Gene Ther.(1999)10:641-648; Ali et al., Hum.Mol.Genet.(1996)5:591-594; International Publication No. 93 / 09239, Srivastava, Samulski et al., J.Vir.(1989)63:3822-3828; Mendelson et al., Virol.(1988)166:154-165; and Flotte et al., Proc.Natl.Acad.Sci.See USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73:7812-7816); retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary cancer virus), but are not limited to these.

[0257] Further expression vectors suitable for use include, but are not limited to, lentiviral vectors, gamma-retroviral vectors, Formy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid virus vectors, and transposon-mediated vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), as well as in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0258] Generally, a suitable vector includes a functional origin of replication, a promoter sequence, a favorable restriction endonuclease site, and one or more selectable markers in at least one organism (e.g., International Publication No. 01 / 96584; International Publication No. 01 / 29058; and U.S. Patent No. 6,326,193).

[0259] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce CAR into immune cells or their precursors (e.g., T cells). Therefore, the expression vector (e.g., a lentiviral vector) of the present invention may contain a nucleic acid encoding CAR. In some embodiments, the expression vector (e.g., a lentiviral vector) includes further elements that assist in the functional expression of the CAR encoded therein. In some embodiments, the expression vector containing the nucleic acid encoding CAR further includes a mammalian promoter. In one embodiment, the vector further includes an elongation factor-1-alpha promoter (EF-1α promoter). The use of the EF-1α promoter may increase the efficiency of expression of downstream transgenes (e.g., nucleic acid sequences encoding CAR). Physiological promoters (e.g., EF-1α promoters) may be less likely to induce genotoxicity mediated by integration and may negate the retroviral vector's ability to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and may be incorporated into the vector of the present invention. In some embodiments, the vector (e.g., a lentiviral vector) further comprises non-essential cis-acting sequences that can improve titer and gene expression. One non-limiting example of a non-essential cis-acting sequence is a central polyprint lactate and central termination sequence (cPPT / CTS) that is important for efficient reverse transcription and nuclear translocation. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vector of the present invention (e.g., a lentiviral vector). In some embodiments, the vector further comprises post-transcriptional regulatory elements. Post-transcriptional regulatory elements can improve RNA translation, improve transgene expression, and stabilize RNA transcripts. An example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vector of the present invention (e.g., a lentiviral vector).The vectors of the present invention may further include further elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long-terminal repeat sequences (LTRs). The terms “long-terminal repeat sequence” or “LTR” refer to a domain of base pairs located at the end of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and functions required for viral replication. In one embodiment, the vector of the present invention (e.g., a lentiviral vector) includes a 3'U3 deletion LTR. Thus, the vector of the present invention (e.g., a lentiviral vector) may include any combination of the elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., a lentiviral vector) may include a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, and a 3'U3 deletion LTR' in addition to the nucleic acid encoding the CAR.

[0260] The vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). Self-inactivating vectors can prevent viral transcription beyond the first round of viral replication. As a result, self-inactivating vectors may be able to infect and subsequently integrate into a host genome (e.g., a mammalian genome) only once and cannot be transmitted further. Therefore, self-inactivating vectors can significantly reduce the risk of producing viruses capable of replication.

[0261] In some embodiments, the nucleic acids of the present invention may be RNA, for example, RNA synthesized in vitro. Methods for the in vitro synthesis of RNA are known to those skilled in the art, and any known method can be used to synthesize RNA containing the CAR encoding sequence of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15:9053. Introducing RNA containing the CAR encoding sequence of the present disclosure into host cells can be carried out in vitro, ex vivo, or in vivo. For example, RNA containing the CAR encoding sequence of the present disclosure can be electroporated into host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) in vitro or ex vivo.

[0262] To evaluate the expression of a polypeptide or a portion thereof, the expression vector introduced into cells may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells intended to be transfected or infected via the viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a simultaneous transfection procedure. To enable expression in host cells, both the selectable marker and reporter genes may be flanked by appropriate regulatory sequences. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0263] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is revealed by some readily detectable characteristic, such as enzymatic activity. Reporter gene expression is evaluated at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82).

[0264] E. Modified immune cells The present invention provides a modified immune cell or precursor (e.g., T cell) comprising a chimeric antigen receptor (CAR) capable of binding IL13Rα2 (e.g., human IL13Rα2), a CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a dominant-negative TGFβII receptor (DN-TGFβRII). The present invention also comprises a modified immune cell or precursor comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0265] One aspect of the present invention includes a modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). The first CAR comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 comprises the amino acid sequence TIFF2026509470000052.tif4157 is included, and HCDR3 is an amino acid sequence Includes TIFF2026509470000053.tif4128. The first CAR also includes a light chain variable region containing three light chain complementarity determining regions (LCDRs). LCDR1 is an amino acid sequence The second CAR contains TIFF2026509470000054.tif4128, LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7) or QHHYSAPWT (SEQ ID NO: 18). The second CAR contains a heavy chain variable region containing three heavy chain complementarity determining regions (HCDRs). HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The second CAR contains TIFF2026509470000055.tif4128, and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27). The second CAR also contains a light chain variable region containing three light chain complementarity determining regions (LCDRs). LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGINLDD (SEQ ID NO: 143) or HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0266] Another aspect of the present invention is, Modified immune cells or their progenitor cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first CAR comprises a heavy chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, and a light chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20. The second CAR includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 or SEQ ID NO: 19 or SEQ ID NO: 8, and a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32 or SEQ ID NO: 9 or SEQ ID NO: 20.

[0267] Modified immune cells or their progenitor cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβII receptor (DN-TGFβRII), wherein the first CAR comprises a single-stranded variable fragment (scFv) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21 or SEQ ID NO: 22, and the second CAR comprises a single-stranded variable fragment (scFv) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 33 or SEQ ID NO: 71. It is also offered.

[0268] Modified immune cells or their progenitor cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), wherein the first CAR contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or 24, and the second CAR contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. It is also offered.

[0269] In one embodiment, the second CAR is wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126YEGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F EGFR isoforms selected from the group consisting of EGFR variant II, or any combination thereof, can be combined.

[0270] In one aspect, the cells are modified T cells. In another aspect, the cells are autologous cells. In another aspect, the cells are autologous cells obtained from a human subject.

[0271] F. Source of immune cells In some embodiments, the source of immune cells (e.g., T cells) is obtained from a subject for ex vivo manipulation. The source of immune cells for ex vivo manipulation may also include, for example, autologous or xenodontic blood, umbilical cord blood, or bone marrow. For example, the source of immune cells may originate from the subject to be treated with the modified immune cells of the present invention, e.g., the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human.

[0272] Immune cells can be obtained from many sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, or lymphoid cells, such as bone marrow cells or lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject being treated, the cells may be allogeneic and / or autologous. The cells are typically primary cells, such as those isolated directly from the subject and / or isolated and frozen from the subject.

[0273] In some embodiments, immune cells are T cells, e.g., CD8+ T cells (e.g., CD8+ naive T cells, central memory T cells, or effector memory T cells), CD4+ T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), stem cell memory T cells, lymphocyte progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), or dendritic cells. In some embodiments, cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In one embodiment, the target cells are induced pluripotent stem (iPS) cells or cells derived from iPS cells, for example, iPS cells that have been generated from a subject and manipulated to modify (e.g., induce mutations in) the expression of one or more target genes, and differentiated into, for example, T cells, e.g., CD8+ T cells (e.g., CD8+ naive T cells, central memory T cells, or effector memory T cells), CD4+ T cells, stem cell memory T cells, lymphocyte progenitor cells, or hematopoietic stem cells.

[0274] In some embodiments, cells include one or more subsets of T cells or other cell types, e.g., the whole T cell population, CD4+ cells, CD8+ cells, and their subpopulations, defined, e.g., function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, e.g., stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, endogenous and adaptive regulatory T (Treg) cells, helper T cells, e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In some embodiments, any number of T cell lines available in the art may be used.

[0275] In some embodiments, the method includes steps of isolating immune cells from a subject, preparing them, processing them, culturing them, and / or manipulating them. In some embodiments, the preparation of manipulated cells includes one or more culture and / or preparation steps. Cells for manipulation as described may be isolated from a sample, e.g., a biological sample, e.g., a sample obtained from or derived from a subject. In some embodiments, the subject from which cells are isolated is a subject having a disease or condition, or requiring cell therapy, or to which cell therapy is administered. In some embodiments, the subject is a human being requiring a specific therapeutic intervention, e.g., adoptive cell therapy for which cells are isolated, processed, and / or manipulated. Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples may be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, including processed samples derived from tissue and organ samples.

[0276] In some contexts, the sample from which cells are derived or isolated is either blood or a blood-derived sample, or a product of or derived from apheresis or leukocyte apheresis. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. Samples may include autologous and allogeneic source samples in the context of cell therapy, such as adoptive cell therapy.

[0277] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a heterogeneous source, such as mice, rats, non-human primates, and pigs. In some embodiments, cell isolation involves one or more preparation and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove undesirable components, concentrate desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0278] In some cases, cells derived from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. The sample, in some aspects, includes lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects, includes cells other than erythrocytes and platelets. In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma fraction and to place the cells into a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in culture medium. In some embodiments, the method includes the preparation of leukocytes from peripheral blood by lysing erythrocytes and density-based cell separation methods such as centrifugation by Percoll or Ficoll gradient.

[0279] In one embodiment, immune cells obtained from the circulating blood of an individual are obtained by apheresis or leukocyte apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells into a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the washing solution may be calcium-deficient, magnesium-deficient, or lack many, if not all, divalent cations for subsequent processing steps. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in a culture medium.

[0280] In some embodiments, the isolation method includes the separation of different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, isolation in some aspects includes, for example, the separation of cells and cell populations based on the expression or expression level of one or more markers, typically antibodies or binding partners that specifically bind to cell surface markers, by incubation, followed generally by a washing step and the separation of cells bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner.

[0281] Such separation steps may be based on positive selection, where cells bound to a reagent are retained for further use, and / or negative selection, where cells that did not bind to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some situations, negative selection may be particularly useful when antibodies that specifically identify cell types are not available in heterogeneous populations, and therefore separation is best performed based on markers expressed by cells other than the desired population. Separation does not need to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or proportion of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or proportion of such cells, but does not need to result in the complete removal of all such cells.

[0282] In some cases, multiple separation steps are performed, in which fractions selected positively or negatively from a single step are subjected to further separation steps, such as subsequent positive or negative selection. In some cases, cells expressing multiple markers simultaneously can be depleted in a single separation step by incubating cells with multiple antibodies or binding partners specific to markers that are targets for negative selection, each of which is a target marker. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0283] In some embodiments, one or more T cell populations are positive for (marker+) or express high levels of one or more specific markers, such as surface markers (markers). hlgh ) cells, or cells that are negative for one or more markers (marker-) or express relatively low levels of (marker low) Cells are either enriched or depleted. For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are absent or expressed at relatively low levels in certain populations of T cells (e.g., non-memory cells), but present or expressed at relatively high levels in certain other populations of T cells (e.g., memory cells). In one embodiment, cells (such as CD8+ cells or T cells, e.g., CD3+ cells) are enriched (i.e., positively selected) with cells that are positive for CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, or expressing them at high surface levels, and / or depleted with cells that are positive for CD45RA, or expressing them at high surface levels (e.g., negatively selected). In some embodiments, cells are enriched or depleted with cells that are positive for CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127), or expressing them at high surface levels. In some examples, CD8+ T cells are enriched with cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28-binding magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0284] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells, such as CD14, B cells, monocytes, or other leukocytes. In some embodiments, CD4+ or CD8+ selection steps are used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further subdivided into subpopulations by positive or negative selection for markers expressed or expressed to relatively high degrees on one or more naive T cell, memory T cell, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based on surface antigens associated with each subpopulation, etc. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, for example, to improve long-term survival, expansion, and / or engraftment after administration, which in some embodiments is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0285] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can enrich or deplete the CD62L-CD8+ and / or CD62L+CD8+ fractions using anti-CD8 antibodies and anti-CD62L antibodies, for example. In some embodiments, the CD4+ T cell population and the CD8+ T cell subpopulation, e.g., central memory (TCM) cells, are enriched. In some embodiments, the enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some embodiments, this is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some cases, isolation of a CD8+ population enriched with TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one case, enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. In some cases, such selections are performed simultaneously, and in others, they are performed sequentially in either order. In some cases, the same CD4 expression-based selection step used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, resulting in the retention of both positive and negative fractions from the CD4-based isolation, which are used in subsequent steps of the method after optionally one or more further positive or negative selection steps.

[0286] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, and CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CDILB, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0287] In some embodiments, cells are incubated and / or cultured before or in combination with genetic engineering. The incubation step may include culture, stimulation, activation, and / or proliferation. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic engineering such as the introduction of recombinant antigen receptors. Conditions may include one or more specific media, temperature, oxygen content, carbon dioxide content, time, active agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulants, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other active agents designed to activate cells. In some embodiments, the stimulating conditions or stimulants include one or more active agents, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the active agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such active ingredients may include antibodies specific to TCR components and / or costimulatory receptors, such as anti-CD3, anti-CD28, and / or one or more cytokines, bound to a solid support such as beads. Optionally, the expansion method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to a culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15, for example, at a concentration of at least about 10 units / mL of IL-2.

[0288] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient. Alternatively, T cells can be isolated from the umbilical cord. In any case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0289] The umbilical cord blood mononuclear cells thus isolated can be used to deplete cells expressing specific antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be achieved using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to physical supports, and antibodies bound to cells.

[0290] The enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. A preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, CD4 by negative selection. + To enrich cells, a monoclonal antibody cocktail typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0291] To isolate a desired population of cells by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In yet another embodiment, more than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml are used. In further embodiments, concentrations of 125 million or 150 million cells / ml may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0292] T cells can also be frozen after the washing step, which does not require a monocyte removal step. While we do not wish to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and, to some extent, monocytes from the cell population. After a washing step to remove plasma and platelets, cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this regard, but in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen at a rate of 1°C per minute to -80°C and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate, uncontrolled freezing at -20°C or in liquid nitrogen, may be used.

[0293] In one embodiment, T cells are included within a population of cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells include a population of T cells. In yet another embodiment, purified T cells include a population of T cells.

[0294] In some embodiments, regulatory T cells (Tregs) can be isolated from a sample. The sample may include, but is not limited to, umbilical cord blood or peripheral blood. In some embodiments, Tregs are isolated by flow cytometry sorting. The sample may be enriched for Tregs before isolation by any means known in the Art. The isolated Tregs may be cryopreserved and / or expanded before use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein by reference in their entirety.

[0295] G. Treatment methods Modified immune cells (e.g., T cells) as described herein may be included in compositions for immunotherapy. The compositions may comprise a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier. A therapeutically effective dose of the pharmaceutical composition containing modified T cells may be administered.

[0296] In one aspect, the present invention encompasses a method for treating a disease or condition in a subject, comprising the step of administering an effective amount of the modified T cells of the present invention to a subject in need. In another aspect, the present invention encompasses a method for treating a disease or condition in a subject, comprising the step of administering a pharmaceutical composition containing an effective amount of the modified T cells of the present invention to a subject in need. In yet another aspect, the present invention encompasses a method for adoptive cell transfer therapy, comprising the step of administering an effective amount of the modified T cells of the present invention to a subject in need.

[0297] Methods for administering immune cells for adoptive cell therapy are publicly known and may be used in connection with the methods and compositions provided. For example, methods for adoptive T cell therapy are described, for example, in Gruenberg et al., U.S. Patent Application Publication No. 2003 / 0170238; Rosenberg, U.S. Patent No. 4,690,915; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; and Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, cell therapy, such as adoptive T-cell therapy, is carried out by autologous transplantation, in which cells are isolated and / or otherwise prepared from a subject to receive cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells originate from a subject in need of treatment, such as a patient, and after isolation and processing, the cells are administered to the same subject.

[0298] In some embodiments, cell therapy, such as adoptive T-cell therapy, is carried out by allogeneic transplantation, in which cells are isolated and / or otherwise prepared from a subject other than the subject to receive or ultimately receive cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0299] In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or condition, such as a tumor, prior to the administration of cells or a cell-containing composition. In some aspects, the subject is refractory or unresponsive to other therapeutic agents. In some embodiments, the subject has a persistent or relapsing disease after treatment with another therapeutic intervention, such as chemotherapy, radiotherapy and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the administration effectively treats the subject despite the subject becoming resistant to other therapies.

[0300] In some embodiments, the subject is responsive to other therapeutic agents, and treatment with these agents reduces the disease burden. In some aspects, the subject is initially responsive to therapeutic agents but shows relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, for example, at high risk of relapse, and therefore cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0301] In some embodiments, the subject has a persistent or relapsing disease after treatment with another therapeutic intervention, such as chemotherapy, radiotherapy, and / or hematopoietic stem cell transplantation (HSCT), including, for example, allogeneic HSCT. In some embodiments, the administration effectively treats the subject despite the subject becoming resistant to another therapy.

[0302] The modified immune cells of the present invention can be administered to animals, preferably mammals, and more preferably humans, to treat cancer. Furthermore, the cells of the present invention can be used to treat any cancer-related condition, particularly for cell-mediated immune responses against tumor cells, where it is desirable to treat or alleviate the disease. The types of cancer that can be treated with the modified cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer. Cancer can be a non-solid tumor (such as a hematological malignancy) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, cancer is a solid tumor or a hematological malignancy. In one embodiment, cancer is a carcinoma. In one aspect, cancer is a sarcoma. In one aspect, cancer is a leukemia. In one aspect, cancer is a solid tumor.

[0303] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, and pheochromocytoma. Examples include sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular carcinoma, seminomas, bladder carcinoma, melanoma, and CNS tumors (gliomas such as brainstem gliomas and mixed gliomas, glioblastoma (also known as glioblastoma pleomorphoni), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, Schwann cell tumor, craniopharyogioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases). In some aspects, the carcinoma is an astrocytoma. In some aspects, the carcinoma is a high-grade astrocytoma.

[0304] Cancers suitable for treatment by the methods disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder carcinoma including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic lung carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma including small cell and non-small cell lung carcinoma, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, intraductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0305] Sarcomas that may be suitable for treatment by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, or other soft tissue sarcomas.

[0306] In one exemplary embodiment, the modified immune cells of the present invention are used to treat myeloma or a myeloma-related condition. Examples of myeloma or a related condition include, but are not limited to, light chain myeloma, nonsecretory myeloma, monoclonal gammaglobulinemia (gamopathy) of unknown significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, the method of the present disclosure is used to treat multiple myeloma. In one embodiment, the method of the present disclosure is used to treat refractory myeloma. In one embodiment, the method of the present disclosure is used to treat relapsed myeloma.

[0307] In one exemplary embodiment, the modified immune cells of the present invention are used to treat melanoma or a melanoma-related condition. Examples of melanoma or a related condition include, but are not limited to, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, achromatic melanoma, or cutaneous melanoma (e.g., cutaneous, ocular, vulvar, vaginal, or rectal melanoma). In one embodiment, the method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, the method of the present disclosure is used to treat refractory melanoma. In one embodiment, the method of the present disclosure is used to treat recurrent melanoma.

[0308] In yet another exemplary embodiment, the modified immune cells of the present invention are used to treat sarcomas or sarcoma-related conditions. Examples of sarcomas or related conditions include, but are not limited to, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumors, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumors, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat liposarcomas such as myxoid / round cell liposarcoma, differentiated / dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, the method of the present disclosure is used to treat myxoid / round cell liposarcoma. In one embodiment, the method of the present disclosure is used to treat refractory sarcoma. In one embodiment, the method of the present disclosure is used to treat recurrent sarcoma.

[0309] The cells of the present invention administered to the patient may be of autologous origin to the patient receiving treatment.

[0310] The administration of the cells of the present invention may be carried out by any convenient method known to those skilled in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, oral ingestion, blood transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously (iv) or via an intraperitoneal route. In other examples, the cells of the present invention may be injected directly into the site of inflammation, the site of local disease, lymph nodes, organs, tumors, etc.

[0311] In some embodiments, cells are administered in a desired dose, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, in some embodiments, the dose of cells is based on the total number of cells (or number per kg of body weight) and a desired ratio of individual populations or subtypes, e.g., CD4+ to CD8+ ratio. In some embodiments, the dose of cells is based on the desired total number of cells (or number per kg of body weight) in individual populations or individual cell types. In some embodiments, the dose is based on a combination of such characteristics, e.g., a desired number of total cells, a desired ratio, and a desired total number of cells in individual populations.

[0312] In some embodiments, CD8 + and CD4 + A population or subtype of cells, such as T cells, is administered in or within a tolerance of a desired dose of total cells, such as a desired dose of T cells. In some cases, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to which the cells are administered, e.g., cells / kg. In some cases, the desired dose is a minimum number of cells or above, or a minimum number of cells per unit body weight or above. In some cases, within the total cells administered at the desired dose, individual populations or subtypes produce a desired ratio (CD4 + vs CD8 + A ratio (or a similar ratio) or a ratio close to it, which exists, for example, within a specific tolerance or error of such a ratio.

[0313] In some embodiments, cells are administered in or within tolerance of one or more desired doses of individual populations or subtypes of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some embodiments, the desired dose is a desired number of cells of a subtype or population, or a desired number of such cells per unit body weight of the subject to which the cells are administered, e.g., cells / kg. In some embodiments, the desired dose is above the minimum or minimum number of cells of a population or subtype, or above the minimum or minimum number of cells of a population or subtype per unit body weight. Thus, in some embodiments, the dose is based on a desired fixed dose and a desired ratio of total cells, and / or on one or more individual subtypes or subpopulations, e.g., each with its desired fixed dose. Thus, in some embodiments, the dose is based on a desired fixed dose or minimum dose of T cells and CD4 + vs CD8 + Based on the desired ratio of cells, and / or CD4 + and / or CD8 + Based on the desired fixed dose or minimum dose of cells.

[0314] In some embodiments, an individual population of cells, or a subtype of cells, is defined as a range of approximately 1 million to approximately 100 billion cells, for example, 1 million to approximately 50 billion cells (for example, approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above values), for example, 10 million to approximately 100 billion cells (for example, approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells) The target is administered cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells (or a range defined by any two of the above values), and in some cases, approximately 100 million to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells), or any value between these ranges.

[0315] In some embodiments, the dose for the whole cell and / or the dose for individual subpopulations of cells is 1 × 10⁻⁶ 5 cells / kg ~ approx. 1×10 11 cells / kg 10 4 Or approximately 1 × 10 5 cells / kg ~ approx. 1×10 11 cells / kg 10 4 From, 10 11 or about 10 11 This is cells / kg body weight, for example, 10 5 ~10 6 The range is cells / kg body weight, for example, 1 × 10⁻⁶ 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1 × 10⁻⁶ 6 cells / kg, or approximately 1 × 10⁶ 5 cells / kg, approximately 1.5×10 5 cells / kg, approximately 2×10 5cells / kg or about 1×10 6 cells / kg. For example, in some embodiments, the cells are 10 4 or about 10 4 to 10 9 or about 10 9 T cells per kilogram (kg) of body weight, such as 10 5 ~10 6 T cells / kg body weight, such as 1×10 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg or 1×10 6 T cells / kg body weight, or about 1×10 5 T cells / kg, about 1.5×10 5 T cells / kg, about  2×10 5 T cells / kg or about 1×10 6 T cells / kg body weight, or are administered within those specific error ranges. In other exemplary embodiments, suitable dosage ranges for the modified cells for use in the methods of the present disclosure are, without limitation, about 1×10 5 cells / kg to about 1×10 6 cells / kg, about 1×10 6 cells / kg to about 1×10 7 cells / kg, about 1×10 7 cells / kg to about 1×10 8 cells / kg, about 1×10 8 cells / kg to about 1×10 9 cells / kg, about 1×10 9 cells / kg to about 1×10 10 cells / kg, about 1×10 10 cells / kg to about 1×10 11 cells / kg. In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 8 cells / kg. In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 7 cells / kg. In other embodiments, a suitable dosage is about 1×10 7 total cells to about 5×10 7 total cells. In some embodiments, a suitable dosage is about 1×10 8 total cells to about 5×108 This refers to a whole cell. In some embodiments, the appropriate dose is approximately 1.4 × 10⁻⁶. 7 The total number of cells is approximately 1.1 × 10⁻⁶. 9 This is a whole cell. In an exemplary embodiment, an appropriate dose for use in the method of this disclosure is approximately 7 × 10⁻⁶ 9 It is the entire cell of an individual.

[0316] In some embodiments, the cell is 10 4 or about 10 4 From, 10 9 or about 10 9 CD4 + and / or CD8 + Cells per kilogram (kg) of body weight, for example, 10 5 ~10 6 CD4 + and / or CD8 + Cells / kg body weight, for example, 1 × 10 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 × 10⁶ 6 CD4 + and / or CD8 + Cells / kg body weight, or approximately 1 × 10⁶ 5 CD4 + and / or CD8 + cells / kg, approximately 1.5×10 5 CD4 + and / or CD8 + cells / kg, approximately 2×10 5 CD4 + and / or CD8 + Cells / kg body weight, or approximately 1 × 10⁶ 6 CD4 + and / or CD8 +Cells are administered at a rate of cells / kg body weight, or within a specific margin of error. In some embodiments, the cells are approximately 1 × 10⁶ 6 Super, about 2.5×10 6 Super, about 5×10 6 Super, about 7.5×10 6 Over or approximately 9 x 10 6 Ultra-individual CD4 + cells, and / or at least about 1 × 10⁻⁶ 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or approximately 9 x 10 6 CD4 + cells, and / or at least about 1 × 10⁻⁶ 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or approximately 9 x 10 6 individual CD8+ cells, and / or at least about 1 × 10⁶ 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or approximately 9 x 10 6 They are administered in the form of individual T cells, or within a specific margin of error. In some embodiments, the cells are approximately 10 8 ~10 12 Or about 10 10 ~10 11 Each T cell, approximately 10 8 ~10 12 Or about 10 10 ~10 11 CD4 + cells, and / or about 10 8 ~10 12 Or about 10 10 ~10 11 CD8 + It is administered to cells, or within a specific margin of error.

[0317] In some embodiments, cells are administered in a desired production ratio or within an acceptable range of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some embodiments, the desired ratio may be a specific ratio or a range of ratios, for example, in some embodiments, the desired ratio (e.g., CD4+) + vs CD8 + The cell ratios range from 5:1 or approximately 5:1 to 5:1 or approximately 5:1 (or greater than approximately 1:5 and less than approximately 5:1), or from 1:3 or approximately 1:3 to 3:1 or approximately 3:1 (or greater than approximately 1:3 and less than approximately 3:1), for example, from 2:1 or approximately 2:1 to 1:5 or approximately 1:5 (or greater than approximately 1:5 and less than approximately 2:1), for example, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1 :1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, or approximately 5:1, approximately 4.5:1, approximately 4:1, approximately 3.5:1, approximately 3:1, approximately 2.5:1, approximately 2:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1 , approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1:1, approximately 1:1.1, approximately 1:1.2, approximately 1:1.3, approximately 1:1.4, approximately 1:1.5, approximately 1:1.6, approximately 1:1.7, approximately 1:1.8, approximately 1:1.9, approximately 1:2, approximately 1:2.5, approximately 1:3, approximately 1:3.5, approximately 1:4, approximately 1:4.5, or approximately 1:5. In some cases, the tolerance is within approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% of the desired ratio, and includes any value between these ranges.

[0318] In some embodiments, the modified cells are administered in single or multiple doses to the subject in need. In some embodiments, the modified cells are administered in multiple doses, for example, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, or once every 4 weeks or every 28 days. In exemplary embodiments, a single dose of the modified cells is administered to the subject in need. In exemplary embodiments, a single dose of the modified cells is administered to the subject in need by rapid intravenous infusion.

[0319] With regard to the prevention or treatment of disease, the appropriate dosage may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, past treatments, the subject's medical history and response to the cells, and the discretion of the attending physician. The composition and cells are administered appropriately to the subject, in some embodiments, either in a single dose or over a series of treatments.

[0320] In some embodiments, cells are administered as part of a combination therapy, concurrently with or sequentially in any order to another therapeutic intervention, such as an antibody or engineered cells or receptors or agents, such as a cytotoxic agent or therapeutic agent. In some embodiments, cells are administered concurrently or sequentially in any order to one or more additional therapeutic agents or in combination with another therapeutic intervention. In some situations, cells are administered concurrently with another therapy that is time-sufficiently close so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered before one or more additional therapeutic agents. In some embodiments, cells are administered after one or more additional therapeutic agents. In some embodiments, one or more additional agents include cytokines such as IL-2 to enhance persistence. In some embodiments, the method includes the administration of chemotherapeutic agents.

[0321] In some embodiments, modified cells of the present invention (e.g., modified cells containing CARs) may be administered to a subject in combination with an immune checkpoint inhibitor. Examples of immune checkpoints include, but are not limited to, CTLA-4, PD-1, and TIM-3. Antibodies may be used to inhibit immune checkpoints (e.g., anti-PD1, anti-CTLA-4, or anti-TIM-3 antibodies). For example, modified cells may be administered in combination with an antibody or antibody fragment that targets, for example, PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, also known as MK-3475) and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or their antigen-binding fragments. In some embodiments, modified cells may be administered in combination with an anti-PD-L1 antibody or its antigen-binding fragment. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In some embodiments, modified cells may be administered in combination with an anti-CTLA-4 antibody or its antigen-binding fragment. Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators, including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems, may also be used. Immune checkpoint modulators may be administered before, after, or concurrently with modified cells containing CARs. In some embodiments, combination therapy including immune checkpoint modulators may enhance the therapeutic effect of the modified cell therapy of the present invention.

[0322] In some embodiments, the biological activity of the manipulated cell population is measured by, for example, one of several known methods after administration of cells. Parameters to be evaluated include the specific binding of manipulated or innate T cells or other immune cells to antigens, in vivo, for example by imaging, or ex vivo, for example by ELISA or flow cytometry. In some embodiments, the ability of manipulated cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009); and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In some embodiments, the biological activity of cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by evaluating clinical outcomes, such as a reduction in tumor volume or tumor burden.

[0323] In some cases, the subject is offered secondary treatment. Secondary treatment includes, but is not limited to, chemotherapy, radiation, surgery, and drug therapy.

[0324] In some embodiments, the subject may undergo conditioning therapy prior to CAR T-cell therapy. In some embodiments, the conditioning therapy includes the step of administering an effective dose of cyclophosphamide to the subject. In some embodiments, the conditioning therapy includes the step of administering an effective dose of fludarabine to the subject. In preferred embodiments, the conditioning therapy includes the step of administering a combination of effective doses of cyclophosphamide and fludarabine to the subject. Administration of conditioning therapy prior to CAR T-cell therapy may enhance the efficacy of CAR T-cell therapy. A method for conditioning a patient for T-cell therapy is described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0325] In some embodiments, certain drug regimens of this disclosure include a lymphocyte depletion step prior to the administration of modified T cells. In exemplary embodiments, the lymphocyte depletion step includes the administration of cyclophosphamide and / or fludarabine.

[0326] In some embodiments, the lymphocyte depletion process involves approximately 200 mg / m². 2 / day~about 2000mg / m 2 / day (for example, 200 mg / m²) 2 / day, 300mg / m 2 / day, or 500mg / m² 2 This includes administration of cyclophosphamide at a dose of ( / day). In exemplary embodiments, the dose of cyclophosphamide is approximately 300 mg / m². 2 This is per day. In some embodiments, the lymphocyte depletion process is approximately 20 mg / m². 2 / day~about 900mg / m 2 / day (for example, 20 mg / m²) 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60mg / m² 2 This includes administration of fludarabine at a dose of ( / day). In an exemplary embodiment, the dose of fludarabine is approximately 30 mg / m². 2 / day

[0327] In some embodiments, the lymphocyte depletion process involves approximately 200 mg / m². 2 / day~about 2000mg / m 2 / day (for example, 200 mg / m²) 2 / day, 300mg / m 2 / day, or 500mg / m² 2 A dose of cyclophosphamide ( / day), and approximately 20 mg / m² 2 / day~about 900mg / m 2 / day (for example, 20 mg / m²) 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60mg / m² 2 The procedure includes the administration of fludarabine at a dose of approximately 300 mg / m² / day. In an exemplary embodiment, the lymphocyte depletion process involves approximately 300 mg / m². 2The daily dose of cyclophosphamide is approximately 30 mg / m². 2 This includes administration of fludarabine at a daily dose.

[0328] In an exemplary embodiment, the dose of cyclophosphamide is 300 mg / m² over 3 days. 2 The dosage is 30 mg / m² / day, and the fludarabine dose is 30 mg / m² over 3 days. 2 / day

[0329] Lymphocyte depletion chemotherapy can be scheduled between day -6 and day -4 (a -1 day window, i.e., administered between day -7 and day -5) in relation to the infusion of T cells (e.g., CAR-T, TCR-T, modified T cells, etc.) on day 0.

[0330] In an exemplary embodiment, for a subject with cancer, the subject receives 300 mg / m² intravenously three days before administration of modified T cells. 2 The subject receives lymphocyte depletion chemotherapy including cyclophosphamide. In an exemplary embodiment, a subject with cancer receives 300 mg / m² intravenously for 3 days prior to the administration of modified T cells. 2 They receive lymphocyte depletion chemotherapy including cyclophosphamide.

[0331] In an exemplary embodiment, for subjects with cancer, the dose is approximately 20 mg / m². 2 / day~about 900mg / m 2 / day (for example, 20 mg / m²) 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60mg / m² 2 The patient receives lymphocyte depletion chemotherapy containing fludarabine at a dose of 30 mg / m² / day. In an exemplary embodiment, for a patient with cancer, the patient receives 30 mg / m² / day. 2 Receive lymphocyte depletion chemotherapy including a dose of fludarabine for 3 days.

[0332] In an exemplary embodiment, for subjects with cancer, the subject receives approximately 200 mg / m². 2 / day~about 2000mg / m 2 / day (for example, 200 mg / m²) 2 / day, 300mg / m 2 / day, or 500mg / m² 2 A dose of cyclophosphamide ( / day), and approximately 20 mg / m² 2 / day~about 900mg / m 2 / day (for example, 20 mg / m²) 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60mg / m² 2 The subject receives lymphocyte depletion chemotherapy containing fludarabine at a dose of approximately 300 mg / m² / day. In an exemplary embodiment, for a subject with cancer, the subject receives approximately 300 mg / m² / day. 2 The daily dose of cyclophosphamide and 30 mg / m² 2 Receive lymphocyte depletion chemotherapy including a dose of fludarabine for 3 days.

[0333] The cells of the present invention may be administered in doses, routes, and time points determined by appropriate preclinical and clinical trials and studies. The cell composition may be administered multiple times in doses within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating a desired disease or condition, as determined by those skilled in the art.

[0334] It is well known in the art that one of the adverse effects following CAR T cell infusion is the development of an immune activation known as cytokine release syndrome (CRS). CRS is an immune activation that results in an increase in inflammatory cytokines. CRS is a known on-target toxicity, and its manifestation is likely to correlate with efficacy. Clinical and experimental measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade ≥ 3 organ toxicity, aggressive clinical intervention, and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leakage, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic increases in cytokines, including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6, have been shown after CAR T cell infusion. One CRS signature is elevated cytokine levels, including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate elevation), and IL-2 (mild elevation). Elevated clinically available inflammatory markers, including ferritin and C-reactive protein (CRP), have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with the expansion of adoptive cells and progressive immune activation. Since patients with high tumor burdens experience more sCRS, it has been demonstrated that the degree of CRS severity is determined by the disease burden at the time of infusion.

[0335] Therefore, the present invention provides an appropriate CRS management strategy for alleviating the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS, without diminishing the antitumor effect of manipulated cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids can be administered to rapidly restore symptoms of sCRS (e.g., grade 3 CRS) without impairing the initial antitumor response.

[0336] In some embodiments, anti-IL-6R antibodies may be administered. One example of an anti-IL-6R antibody is tocilizumab, a monoclonal antibody approved by the Food and Drug Administration, also known as atlizumab (marketed as Actemra or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated nearly immediate reversal of CRS.

[0337] CRS is generally managed based on the observed severity of the syndrome, and interventions are adjusted accordingly. Decisions regarding CRS management may be based not only on clinical laboratory values, but also on clinical signs and symptoms, as well as the response to interventions.

[0338] Mild to moderate cases are generally treated with fluid therapy and symptom management with nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability, in which case administration of tocilizumab is recommended. First-line management of CRS may, in some aspects, be tocilizumab at a indicated dose of 8 mg / kg IV over 60 minutes (not exceeding 800 mg / dose); tocilizumab may be repeated every Q8 hours. In the event of a suboptimal response to the initial dose of tocilizumab, further doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms, insufficient clinical improvement within 12–18 hours, or an inadequate response to tocilizumab may be treated with high-dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1–2 mg / kg. Patients with more severe hemodynamic instability or more severe respiratory symptoms may be given high-dose corticosteroid therapy early in the course of CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0339] Features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy, consistent with the clinical manifestations of CRS (Henter, 2007). MAS appears to be a response to the immune activation resulting from CRS and should therefore be considered a symptomatic manifestation of CRS. MAS is similar to HLH (also a response to immune stimulation). The clinical syndrome of MAS is characterized by high-grade non-relieving fever, cytopenia affecting at least two of the three blood systems, and hepatosplenomegaly. This is associated with high serum ferritin, soluble interleukin-2 receptors and triglycerides, and decreased circulating natural killer (NK) activity.

[0340] Modified immune cells containing CARs of the present invention may be used in therapeutic methods described herein. In one aspect, the present invention includes a method for treating cancer in a subject in need, comprising the step of administering any one of the modified immune cells or progenitor cells disclosed herein to the subject. A further aspect of the present invention includes a method for treating cancer in a subject in need, comprising the step of administering modified immune cells or progenitor cells produced by any one of the methods disclosed herein to the subject.

[0341] One aspect of the present invention provides a method for treating glioblastoma in subjects where such treatment is needed. The method comprises administering an effective amount of modified T cells to a subject, comprising a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain capable of binding IL13Rα2, a second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII).

[0342] In one aspect, the first CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains TIFF2026509470000056.tif4160 and HCDR3 has an amino acid sequence Heavy chain variable region, including TIFF2026509470000057.tif4128, and / or A light chain variable region containing three light chain complementarity-determining regions (LCDRs), wherein LCDR1 is an amino acid sequence Light chain variable region containing TIFF2026509470000058.tif4130, where LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7) or QHHYSAPWT (SEQ ID NO: 18). Includes.

[0343] In one embodiment, the second CAR is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence A heavy chain variable region containing TIFF2026509470000059.tif4128 and in which HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), and / or A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30). Includes.

[0344] Another aspect of the present invention is, A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells to the subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and DN-TGFβRII, wherein the first CAR comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20, and the second CAR comprises SEQ ID NO: A method comprising a heavy chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region containing an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32. To provide.

[0345] Another aspect of the present invention is, A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and DN-TGFβRII to the subject, wherein the first CAR comprises a single-stranded variable fragment (scFv) having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21 or SEQ ID NO: 22, and the second CAR comprises an scFv having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, 44, or 142, It includes.

[0346] Another aspect of the present invention is, A method for treating glioblastoma in a subject in need, comprising the step of administering an effective amount of modified T cells comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and DN-TGFβRII to the subject, wherein the first CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 55 or SEQ ID NO: 56, and the second CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36 or 197, To provide.

[0347] In one embodiment, the second CAR includes a heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 144 or SEQ ID NO: 145, and a light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 146 or SEQ ID NO: 147.

[0348] In one embodiment, DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0349] H. Expansion of immune cells Whether before or after cell modification to express CAR, cells are, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; They can be activated and proliferated using methods such as those described in U.S. Patent Application Publication No. 20060121005. For example, the T cells of the present invention can be proliferated by contact with a surface to which an agent stimulating CD3 / TCR complex-related signaling and a ligand stimulating a co-stimulatory molecule on the surface of the T cell are attached. In particular, the T cell population can be stimulated by contact with an anti-CD3 antibody or its antigen-binding fragment, or with an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co-stimulation of accessory molecules on the surface of the T cell, ligands that bind to the accessory molecules are used. For example, T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention in the same manner as other methods and reagents known in the art (see, for example, ten Berge et al., Transplant Proc. (1998) 30(8):3975-3977; Haanen et al., J.Exp.Med. (1999) 190(9):1319-1328; and Garland et al., J.Immunol.Methods (1999) 227(1-2):53-63).

[0350] T cells expanding by the methods disclosed herein can be increased to approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000 or more, and any whole integer or subinteger in between. In one embodiment, T cells expand in the range of approximately 20 to approximately 50 times.

[0351] After culturing, the T cells can be incubated in cell medium within the culture device for a certain period, or until the cells reach confluence or high cell density, for optimal subculturing before transferring the cells to another culture device. The culture device can be any culture device commonly used for culturing cells in vitro. Preferably, the confluence level is 70% or higher before transferring the cells to another culture device. More preferably, the confluence level is 90% or higher. The period can be any time suitable for culturing cells in vitro. The T cell medium can be changed at any time during T cell culture. Preferably, the T cell medium is changed approximately every 2-3 days. The T cells are then harvested from the culture device and can then be used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes the step of cryopreserving expanded T cells. The cryopreserved T cells are thawed before nucleic acids are introduced into the T cells.

[0352] In another embodiment, the method comprises the steps of isolating T cells and expanding T cells. In yet another embodiment, the present invention further comprises the step of cryopreserving T cells before expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding chimeric membrane proteins.

[0353] Another method for ex vivo cell expansion is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 may be an alternative to or addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells involves the addition of cell growth factors as described in U.S. Patent No. 5,199,942, or other factors such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding T cells involves the step of culturing T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0354] The culture steps described herein (after contact with the agent or electroporation described herein) may be very short, for example less than 24 hours, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture steps described herein further (after contact with the agent described herein) may be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more.

[0355] Various terms are used to describe cells in culture. A cell culture generally refers to cells taken from a living organism and grown under controlled conditions. A primary cell culture is a culture of cells, tissue, or organ taken directly from an organism and before the first subculturing. Cells are expanded under culture conditions when placed in a growth medium under conditions that promote cell proliferation and / or division, resulting in a larger population of cells. When cells are expanded under culture conditions, the rate of cell proliferation is typically measured by the amount of time it takes for the cells to double in number, also known as doubling time.

[0356] Each passage is called a passage. When cells are passaged, they are referred to as having been passaged. A particular cell population or cell line may be named or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. A primary culture, i.e., the first culture after cells have been isolated from tissue, is designated as P0. After the first passage, the cells are represented as a secondary culture (P1 or passage 1). After the second passage, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that many population doublings can occur during the passage period; therefore, the number of population doublings in a culture is greater than the number of passages. The expansion of cells during the interpassage period (i.e., the number of population doublings) depends on many factors, including but not limited to seeding density, substrate, medium, and interpassage time.

[0357] In one embodiment, cells may be cultured for several hours (approximately 3 hours) to approximately 14 days or any integer value in any unit of time in between. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human fetal serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta and TNF-α, or any other additives for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, and are either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines for T cell proliferation and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the culture of cells injected into the target. Target cells are maintained under conditions necessary to support proliferation, for example, at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0358] The culture medium used to culture T cells may contain an active agent that can co-stimulate the T cells. For example, an active agent that can stimulate CD3 is an antibody against CD3, and an active agent that can stimulate CD28 is an antibody against CD28. Cells isolated by the methods disclosed herein can be magnified by about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 900x, 10,000x, 100,000x, 1,000,000x, 10,000,000x or more. In one embodiment, T cells are enlarged by approximately 20 to 50 times or more. In one embodiment, human regulatory T cells are enlarged via KT64.86 artificial antigen-presenting cells (aAPCs) coated with anti-CD3 antibody. Methods for enlarging and activating T cells can be found in U.S. Patents 7,754,482, 8,722,400 and 9,555,105, the contents of which are incorporated herein by reference in their entirety.

[0359] In one embodiment, a method for expanding T cells may further include a step of isolating the expanded T cells for further use. In another embodiment, a method for expanding T cells may further include subsequent electroporation and subsequent culture of the expanded T cells. The subsequent electroporation may include a step of introducing nucleic acids encoding an active agent into an expanded population of T cells, such as a step of transducing the expanded T cells, a step of transfecting the expanded T cells, or a step of electroporating nucleic acids into the expanded T cells, the active agent further stimulating the T cells. The active agent may stimulate the T cells by stimulating further expansion, effector function, or another T cell function.

[0360] I. Methods for producing genetically modified immune cells This disclosure provides a method for producing or generating modified immune cells or their precursors (e.g., T cells) of the present invention for tumor immunotherapy, such as adoptive immunotherapy.

[0361] In some embodiments, CARs are introduced into cells by expression vectors. Expression vectors comprising nucleic acid sequences encoding the CARs of the present invention are provided herein. Suitable expression vectors include, but are not limited to, lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, transposon-mediated vectors such as Sleeping Beauty and Piggybak, and naked DNA containing integrases such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0362] In one embodiment, the nucleic acid encoding the CAR is introduced into a cell via viral transduction. In one embodiment, viral transduction involves contacting an immune cell or progenitor cell with a viral vector containing the nucleic acid encoding the CAR. In one embodiment, the viral vector is an adeno-associated virus (AAV) vector. In one embodiment, the AAV vector contains 5'ITR and 3'ITR derived from AAV6. In one embodiment, the AAV vector contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In one embodiment, the AAV vector contains a polyadenylated (Poly-A) sequence. In one embodiment, the Poly-A sequence is a bovine growth hormone (BGH) Poly-A sequence.

[0363] Adenovirus expression vectors are based on adenoviruses that have low integration into genomic DNA but high efficiency in transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to (a) support the packaging of the expression vector and (b) ultimately express CAR in host cells. In some embodiments, the adenovirus genome is a 36kb linear double-stranded DNA, and foreign DNA sequences (e.g., nucleic acids encoding CAR) may be inserted to replace large fragments of the adenovirus DNA in order to construct the expression vector of the present invention (see, for example, Danthinne and Imperiale, Gene Therapy (2000) 7(20):1707-1714).

[0364] Another expression vector is based on adeno-associated virus (AAV) utilizing an adenovirus coupled system. This AAV expression vector has a high frequency of integration into the host genome. AAV expression vectors can infect non-dividing cells and are therefore useful for gene delivery to mammalian cells, for example, in tissue culture or in vivo. AAV vectors have a broad host range in terms of infectivity. Details regarding the production and use of AAV vectors are described in U.S. Patents No. 5,139,941 and No. 4,797,368.

[0365] Retroviral expression vectors can be integrated into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and packaged into specific cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding CARs) into the viral genome at specific locations to produce a virus that is replication-deficient. While retroviral vectors can infect a wide variety of cell types, CAR integration and stable expression require host cell division.

[0366] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses containing other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env (see, for example, U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, making the vector biologically safe. Lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression, for example, of CAR-encoding nucleic acids, both in vivo and ex vivo (see, for example, U.S. Patent 5,994,136).

[0367] An expression vector containing the nucleic acid of this disclosure can be introduced into host cells by any means known to those skilled in the art. The expression vector may optionally contain a viral sequence for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, gene gun, transfection, lipofection, etc. Host cells may be grown and expanded in culture before introduction of the expression vector, and then subjected to appropriate treatment for introduction and integration of the vector. The host cells may then be expanded and screened for markers present in the vector. Various markers that may be used are known in the art and may include HPRT, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, host cells, immune cells or their precursors, e.g., T cells, NK cells, or NKT cells.

[0368] The present invention also provides genetically modified cells that stably express the CARs of this disclosure. In some embodiments, the genetically modified cells are genetically modified T lymphocytes (T cells), naive T cells (TNs), memory T cells (e.g., central memory T cells (TCMs), effector memory cells (TEMs)), natural killer cells (NK cells), and macrophages that can produce therapeutically appropriate offspring. In some embodiments, the genetically modified cells are autologous cells. In some embodiments, the modified cells are resistant to T cell exhaustion.

[0369] Modified cells (e.g., including CARs) can be produced by stably transfecting host cells with an expression vector containing the nucleic acids of the Disclosure. Further methods for generating the modified cells of the Disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrophoresis transfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection and / or magnetofection using gene guns). Transfected cells expressing the CARs of the Disclosure can be expanded ex vivo.

[0370] Physical methods for introducing expression vectors into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing expression vectors into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0371] Suitable lipids for use can be obtained from commercial suppliers. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid storage solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform can be used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. Multilayer liposomes spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). Compositions that have structures different from the usual vesicle structure in solution are also included. For example, lipids can take on micelle structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also conceivable.

[0372] Various assays can be performed to confirm the presence of nucleic acids in host cells, regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to other inhibitors of the present invention. Such assays include molecular biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; and biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting) or by assays described herein for identifying active ingredients within the scope of the present invention.

[0373] In one embodiment, the nucleic acid introduced into the host cell is RNA. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA can be produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Using appropriate primers and RNA polymerase, PCR can directly convert DNA of interest from any source into a template for in vitro mRNA synthesis. The source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source.

[0374] PCR may be used to create a template for in vitro transcription of mRNA that will subsequently be introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region substantially complementary to the region of DNA used as the template for PCR. "Substantially complementary," as used herein, refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary. A substantially complementary sequence can anneal to or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, a primer can be designed to amplify a portion of a gene that is normally transcribed in cells (an open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene that codes for a specific domain of interest. In one embodiment, a primer is designed to amplify a coding region of human cDNA including all or part of the 5' and 3' UTRs. Primers useful for PCR are produced by synthetic methods well known in the art. A "forward primer" is a primer containing a region of nucleotides substantially complementary to the nucleotides on the DNA template located upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5 position relative to the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer containing a region of nucleotides substantially complementary to the double-stranded DNA template located downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position relative to the DNA sequence to be amplified relative to the coding strand.

[0375] Chemical structures that have the ability to enhance RNA stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3000 nucleotides long. The lengths of the 5' and 3' UTR sequences appended to the coding region can be modified by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the lengths of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0376] The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the gene of interest. Alternatively, non-endogenous UTR sequences can be added to the gene of interest by incorporating UTR sequences into forward and reverse primers, or by any other modification of the template. The use of non-endogenous UTR sequences for the gene of interest may be useful to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, based on UTR properties known in the art, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA.

[0377] In one embodiment, the 5'UTR may contain the Kosack sequence of an endogenous gene. Alternatively, if a non-endogenous 5'UTR has been added to the gene of interest by PCR as described above, the consensus Kosack sequence can be redesigned by adding the 5'UTR sequence. While the Kosack sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to enable efficient translation. The need for Kosack sequences for many mRNAs is well known in the art. In another embodiment, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in cells. In yet another embodiment, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of mRNA.

[0378] To enable RNA synthesis from a DNA template without requiring gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence acting as a promoter for RNA polymerase is added to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter, as described elsewhere in this specification. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

[0379] In one embodiment, mRNA has both a 5' cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability within the cell. In circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemer products that are unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end results in normal-sized mRNA that is not effective for eukaryotic transfection, even if polyadenylated after transcription.

[0380] In linear DNA templates, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur.J.Biochem., 270:1485-65 (2003)).

[0381] Poly(A) tails can be prepared during PCR by using reverse primers containing poly(T) tails, such as 100T tails (sizes can range from 50 to 5000T), or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNA and reduce RNA degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail consists of 100 to 5000 adenosine molecules.

[0382] The poly(A) tail of RNA can be further elongated after in vitro transcription using a poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides approximately doubles the RNA translation efficiency. Furthermore, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

[0383] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is known in the art and is provided using the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0384] In some embodiments, RNA, such as in vitro transcribed RNA, is electropermeated into cells. Any solute suitable for cell electropermeation may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0385] In some embodiments, the nucleic acid encoding the CAR in this disclosure is RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art, and any known method can be used to synthesize RNA containing the CAR encoding sequence. Methods for introducing RNA into host cells are known in the art; see, for example, Zhao et al. Cancer Res. (2010) 15:9053. Introducing RNA containing the CAR encoding nucleotide sequence into host cells can be carried out in vitro, ex vivo, or in vivo. For example, RNA containing the CAR encoding nucleotide sequence can be electroporated into host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) in vitro or ex vivo.

[0386] The disclosed methods can be applied to the modulation of T cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infectious diseases, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0387] This method also offers the ability to regulate expression levels over a wide range by, for example, altering the amount of promoter or input RNA, enabling individual control of expression levels. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0388] One advantage of the RNA transfection method of the present invention is that RNA transfection is inherently transient and does not require a vector. The RNA transgene can be delivered to lymphocytes as a minimal expression cassette without requiring additional viral sequences, and expressed therein after a short in vitro cell activation. Under these conditions, the likelihood of the transgene being integrated into the host cell genome is low. Due to the efficiency of RNA transfection and its ability to uniformly modify an entire population of lymphocytes, cell cloning is not required.

[0389] Genetic modification of T cells using in vitro transcribed RNA (IVT-RNA) employs two different strategies, both of which have been successfully tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. To achieve long-term expression of the introduced IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0390] Several IVT vectors are known in the literature that have been genetically modified to produce stabilized RNA transcripts and are used in a standardized manner as templates for in vitro transcription. Currently, protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest with an untranslated region (UTR) adjacent to either the 3' and / or 5' ends, and a 3' polyadenylic cassette containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylic cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylic cassette corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzymatic cleavage site after linearization, extending or shielding the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs.

[0391] In another context, RNA constructs are delivered to cells by electroporation. See, for example, the designs and methodologies for electroporation of nucleic acid constructs into mammalian cells, as taught in U.S. Patent Publication Nos. 2004 / 0014645, 2005 / 0052630, 2005 / 0070841, 2004 / 0059285, and 2004 / 0092907. Various parameters, including the electric field strength required for electroporation of any known cell type, are publicly known in the relevant research literature and numerous patents and applications in the art. See, for example, U.S. Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Apparatus for the therapeutic application of electroporation, such as the MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), is commercially available and described in U.S. patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223; U.S. Patent No. 5,993,434; U.S. Patent No. 6,181,964; U.S. Patent No. 6,241,701; and U.S. Patent No. 6,233,482; electroporation can also be used for in vitro cell transfection, as described, for example, in U.S. Patent Application Publication No. 20070128708. Electroporation can also be used to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated delivery of nucleic acids, including expression constructs, into cells using any of the many available apparatuses and electroporation systems known to those skilled in the art presents an exciting new means for delivering RNA of interest to target cells.

[0392] J. Pharmaceutical compositions and formulations The present invention also provides populations of immune cells, compositions containing and / or concentrated with such cells, for example, compositions in which cells expressing CAR constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells or specific types of cells such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Therapeutic methods for targeting and administering cells and compositions, for example to a patient, are also provided.

[0393] The pharmaceutical compositions and formulations also include compositions containing cells for administration, for example, compositions in unit dose form containing a given number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally contain one or more arbitrary pharmaceutically acceptable carriers or excipients. In some embodiments, the composition contains at least one further therapeutic agent.

[0394] The term "pharmaceutical preparation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain any further ingredients that are unacceptably toxic to the subject to which the preparation is administered. "Pharmacovigilant carriers" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subject. Pharmacovigilant carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some cases, the choice of carrier is determined, in part, by the specific cells and / or the method of administration. Thus, a variety of suitable preparations exist. For example, a pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some cases, mixtures of two or more preservatives are used. Preservatives or mixtures thereof are typically present in amounts of about 0.0001% to about 2% by weight of the total composition. The carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, alkylparabens such as butyl or benzyl alcohol, methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0395] In some cases, buffers are included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some cases, mixtures of two or more buffers are used. The buffer or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known. Exemplary methods are described in detail, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0396] The formulation may include an aqueous solution. The formulation or composition may also contain multiple active ingredients useful for a specific indication, disease, or condition being treated with cells, preferably those having complementary activity to cells, provided that their respective activities do not adversely affect each other. Such active ingredients are appropriately present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. In some embodiments, the pharmaceutical composition contains cells in an amount effective to treat or prevent a disease or condition, such as a therapeutic or prophylactic dose. The therapeutic or prophylactic efficacy in some embodiments is observed by periodic evaluation of the subject being treated. The desired dose can be delivered by a single bolus of cells, by multiple bolus of cells, or by continuous infusion of cells.

[0397] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, oral, sublingual, or suppository administration. In some embodiments, cell populations are administered parenterally. As used herein, the term “parenteral” includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, cells are administered to subjects by peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH in some aspects. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline solution, phosphate-buffered saline solution, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), or suitable mixtures thereof.

[0398] Sterile injection solutions can be prepared by incorporating cells into a solvent, for example, by mixing them with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, or dextrose. Depending on the desired administration route and preparation, the composition may contain auxiliary substances such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling or thickening agents, preservatives, flavoring agents, and / or coloring agents. In some cases, appropriate preparations can be prepared by referring to standard texts.

[0399] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of microbial action can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Sustained absorption of the pharmaceutical form for injection can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0400] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0401] The contents of papers, patents, and patent applications, as well as all other documents and electronically available information referred to or cited herein, are incorporated herein by reference in whole, to the same extent that each individual publication is indicated to be incorporated by specific and individual reference. The applicant reserves the right to physically incorporate into this application any and all material and information from such papers, patents, patent applications, or other physical and electronic documents.

[0402] While the present invention has been described with reference to certain embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications can be made to adapt specific circumstances, materials, substance compositions, processes, process steps, or steps to the object, spirit, and scope of the invention. All such modifications are intended to fall within the scope of the appended claims. While certain embodiments have been described in detail here, this will be better understood by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the invention. [Examples]

[0403] Experimental example The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but rather encompasses all variations that become apparent as a result of the teachings provided herein.

[0404] material and method Cell lines and cultures: Human GSC cell line (5077) was derived from excised tumor tissue obtained from the University of Pennsylvania Institutional Review Board, along with written informed consent from the patient (Department of Neurosurgery, Perelman School of Medicine, Philadelphia, PA). Human GSC cell line (5077) was maintained in DMEM F12 Ham supplemented with penicillin / streptomycin, GlutaMAX-1, B27-A, epidermal growth factor, and basic fibroblast growth factor (Corning, Corning, NY). U87MG cell line (ATCC HTB-14) was obtained from the American Type Culture Collection and cultured in MEM supplemented with 10% fetal bovine serum (FBS) containing GlutaMAX-1, HEPES, Pilbert, and penicillin / streptomycin (Thermo Fisher Scientific, Carlsbad, CA). This cell line was engineered to express EGFRvIII protein, click beetle green luciferase, and green fluorescent protein through single-cell purification and expansion. The PC3 prostate cancer cell line (CRL-1435) was obtained from ATCC and cultured in D10 medium consisting of DMEM supplemented with 10% FBS, HEPES, penicillin, and streptomycin. D270 glioma cells were grown and passaged in the flanks of NSG mice. The human glioma cell line U251 was provided by Dr. Jay Dorsey (Department of Radiation Oncology, University of Pennsylvania). Cells were routinely screened for identity and mycoplasma contamination.

[0405] Immunohistochemical staining: Immunohistochemical staining was performed on tissue sections derived from NSG mice using recombinant anti-TGFβ1 antibody (ab215715, Abcam, Waltham, Boston) and DAPI. These mice were intracranially implanted with U87 and D270 gliomas, a procedure facilitated by Daniel Martinez (Pathology Core Laboratory of the Children's Hospital of Philadelphia Research Institute, PA). Tissue sections of the mouse spleen and cerebral cortex were used as positive and negative controls, respectively.

[0406] Vector constructs: The 806-Hu07-mCherry CAR was assembled by combining EGFR-targeting scFv(806) and IL13Rα2-targeting scFv(Hu07), which were synthesized and ligated in a pTRPE lentiviral vector with the EF1 alpha promoter. The construct was terminated at mCherry (Twist Bioscience, San Francisco, CA). The dnTGFβRII structure was digested using AvrII and SalI enzymes and ligated into the 806-Hu07-mCherry construct at the same enzymatic site, replacing the mCherry gene to produce the 806-Hu07-dnTGFβRII CAR construct. CAR-dnTGFβRII-M5 was provided by Joseph A. Fraietta of Perelman School of Medicine, University of Pennsylvania. The control CAR-CD19 was provided by Carl H. June's laboratory, University of Pennsylvania. All CARs have the same second-generation CAR structure, except for the scFv component.

[0407] In vitro transduction and culture of human T cells: Lentiviruses were packaged in HEK 293 T cells using a split genome approach, and titers were determined using SUPT1 cells (CRL-1942) obtained from ATCC. Normal human T cells were isolated from PBMCs at the Human Immunology Core of the University of Pennsylvania and transduced using lentiviral vectors. T cells were stimulated for 5 days with Dynabeads Human T-Activator CD3 / CD28 (Life Technologies, Carlsbad, CA) at a 3:1 bead-to-cell ratio. Cell concentration was determined using a Coulter Multisizer (Beckman Coulter, Brea, CA), and the cells were refueled at a volume of approximately 300 fl until completely quiescent (0.7 × 10⁶). 6 Cells were maintained at a concentration of cells / mL. Cells were cultured in R10 medium (RPMI-1640 supplemented with GlutaMAX-1, HEPES, Pilbert, penicillin / streptomycin and 10% FBS) containing 30 IU / mL of rhIL-2 (Thermo Fisher Scientific, Carlsbad, CA). CAR T cells were then cryopreserved in a mixture of 90% FBS and 10% DMSO for future use.

[0408] TGF-β ELISA: Tumor cell lines were cultured for 3 days at a density of 3e6 cells per flask. The culture supernatant was collected by centrifugation and stored at -80°C. Culture media containing 10% fetal bovine serum are expected to have excess TGF-β1 secretion. Therefore, the control medium should be used as a blank and subtracted from the sample. The thawed culture supernatant was processed using the TGF-β1 ELISA kit (R&D, DY240-05), and the sample was diluted 4-fold before activation of potential TGF-β1 into an immunoreactive form.

[0409] Bioluminescent cytotoxicity assay: U87vIII-CBG luciferase target cells were co-cultured with effector T cells for 16 hours at different effector:target ratios using either 20 ng / ml hTGF-β1 or medium alone. Before measuring luminescence, 15 μg of D-luciferin (Gold Biotechnology, St. Louis, MO) was added and incubated at room temperature for 10 minutes. Luminescence was measured using a BioTek Synergy H4 hybrid multimode microplate reader. Calculations of cytotoxic results established that target cells alone exhibited 0% lysis, with maximum lysis observed upon treatment with 5% SDS solution (Thermo Fisher Scientific, Carlsbad, CA).

[0410] Growth assay: U87vIII cells were irradiated at 10,000 rad for 40 minutes. Subsequently, 0.2e6 tumor cells and 1e6 T cells were co-cultured in sets of three in 4 ml of R10 medium supplemented with either 20 ng / ml human TGF-β1 or medium alone in 12-well plates. When the medium turned yellow around day 4 or 5, it was replenished. On days 7, 14, and 21, 1–2 ml of supernatant was collected from each well, centrifuged, and stored at -80°C for subsequent cytokine analysis (Eve Technologies, Calgary, Canada). The T cells were then resuspended and counted using a Beckman Coulter Multisizer 3 Cell Counter. The 1e6 T cells were transferred to new 0.2e6 irradiated U87vIII cells, and growth assays were continued weekly, with remaining T cells stained for phenotypic and CAR expression.

[0411] Impedance cytotoxicity assay: Target tumor cells of 2e5 were seeded in Axion Biosystems microelectrode-containing 96-well plates (Axion Biosystems, Atlanta, GA). Prior to the experiment, each impedance plate was prepared by coating with 20 μg / mL laminin overnight at 37°C. After coating was complete, the wells were rinsed three times with diH2O, and then 100 μL of cell culture medium was placed on top. The plates were placed in an Axion Biosystems ZHT analyzer (Axion Biosystems, Atlanta, GA) and baseline readings of background impedance in the absence of cells were recorded. After baseline establishment, the plates were removed from the analyzer and 5e5 target cells were seeded at a volume of 200 μL / well. After seeding the cells on the plates, the plates were left in a cell culture hood at room temperature for 1 hour to ensure sedimentation and adhesion of cells to the microelectrode on the bottom surface. The plates were then returned to the analyzer and data acquisition was started. For cell monolayer proliferation measurements, data were collected every minute for 24 hours. For cytotoxicity assessment, the instrument was paused for 24 hours and the medium was replaced with a medium containing a 1:1 dose of effector cells or UTD control T cells, or medium only. Changes in impedance are reported as the resistance component of the complex impedance, as previously described. Using AxIS Z software (Axion Biosystems, Atlanta, GA), all data were corrected for “medium only” to remove small changes in medium-only impedance over time, and then normalized to the impedance at the time of effector cell addition. % cell lysis calculations utilize untreated controls and complete lysis controls to determine the % of target cell lysis, as follows. TIFF2026509470000060.tif13128

[0412] Flow cytometry: A 5-laser LSR Fortessa flow cytometer was used for analysis. First, cells were stained with a viability stain in PBS (Thermo Fisher Scientific, Carlsbad, CA). Then, cells were stained with appropriate antibodies in FACS buffer (0.5% BSA in PBS) for 30 minutes at 4°C. CAR expression was detected using biotinylated protein L (GenScript) antibody and streptavidin-conjugated PE (BD Biosciences). PE anti-human TGF-β receptor II antibody (399703) was used to detect dnTGFβRII expression on CAR. BD Phosflow antibody (562586) was used to detect the phosphorylated SMAD2 / 3 ability of CAR T cells. CD69-FITC (clone FN50, BioLegend) and CD25-PerCP / cyanine 5.5 (clone BC96, BioLegend) were used to detect T cell activation.

[0413] Co-culture assay: In co-culture experiments, transduced T cells or UTD T cells (2e5 cells per well in 100 μL of R10 medium) were co-cultured with target cells (2e5 cells per well in 100 μL of R10 medium) in 96-well round-bottom tissue culture plates at 37°C and 5% CO2 for 16 or 20 hours. In PD-1 marker staining assays, transduced T cells or UTD T cells were distinguished by viability staining (Thermo Fisher Scientific, Carlsbad, CA), followed by human CD3 (clone OKT3, BioLegend, San Diego, CA) staining. BV711-conjugated anti-human PD-1 (clone EH12.2H7, BioLegend, San Diego, CA) was used to detect PD-1 expression. In the T cell phenotyping assay, transduced T cells or UTD T cells (5e5 cells per well in 500 μL of R10 medium) were co-cultured with target cells (2.5e5 cells per well in 500 μL of R10 medium) in a 48-well flat-bottom tissue culture plate. Prior to co-culture with T cells, the target cells were irradiated at 10,000 rad. On days 2 and 4, 2.5e5 irradiated target cells were added to each well. After 5 days of co-culture, 500 μl of supernatant was removed from each well, gently resuspended, and 250 μl of cells were transferred to a 96-well round-bottom plate for staining. Human T cells were distinguished by viability staining (Thermo Fisher Scientific, Carlsbad, CA), followed by human CD3 and CD8 staining (clonal OKT3 and clone SK1, BioLegend, San Diego, CA). To detect T cell phenotypes, BV711-conjugated anti-human CD45RA (clone HI100, BioLegend, San Diego, CA) and APC-conjugated anti-human CCR7 (clone G043H7, BioLegend, San Diego, CA) were used. Staining was appropriately controlled using isotype antibodies. Cells were washed twice with PBS (FACS buffer) containing 2% fetal bovine serum before and after each staining. Fluorescence was assessed using a BD LSRFortessa flow cytometer, and data were analyzed with FlowJo software.

[0414] Mouse model: All mouse experiments were conducted according to protocols approved by the Animal Experiments Committee (IACUC). In the orthotopic tumor model, 5 × 10⁶ mice were introduced into 6-8 week old NSG mice. 5 Transplant individual U87MG-CBG-GFP cells intracranially, or 5 × 10⁶ 5 Individual D270MG-CBG-GFP cells were subcutaneously transplanted. Intracranial surgical transplantation was performed using a stereotactic neurosurgical device, and tumor cells were transplanted 2 mm to the right and 2 mm anterior to the sarcoma suture, and 2 mm into the brain. In the subcutaneous model, 5 × 10¹⁶ cells were transplanted into NSG mice on day 0. 5 Individual D270 tumors were subcutaneously injected in 100 μL of PBS. Following intraperitoneal injection of D-luciferin (Gold Biotechnology, St. Louis, MO) according to the manufacturer's instructions, tumor progression was assessed by luminescence emission using the Xenogen IVIS spectrum. During the experiment, the length and width of the subcutaneous tumors were measured using calipers. Tumor size was calculated as the tumor area by multiplying the two dimensions. Seven–eight days after tumor transplantation, T cells were intravenously injected via the tail vein in a total volume of 100 μL of PBS. Survival was tracked over time until predetermined endpoints approved by IACUC were reached.

[0415] Statistical analysis: Data are presented as mean ± SEM. RNA-seq data from TCGA were analyzed using the Kruskal-Wallis test. Growth assays were analyzed using unpaired t-tests. Impedance and cytolysis assays were analyzed using standard one-way ANOVA and Tukey's test to compare differences between groups. Flow experiments were analyzed using two-way ANOVA and Tukey's test to compare differences between groups. Survival curves were analyzed using Kaplan-Meier (log-rank test). For in vivo tumor studies, linear regression was used to test for significant differences between experimental groups. Survival times were plotted using Kaplan-Meier curves based on time to the end of the experiment. All statistical analyses were performed using Prism software version 9 (GraphPad, La Jolla, CA).

[0416] Example 1: Armored nicistronic CAR T cells with dominant-negative TGF-β receptor II to mitigate antigen heterogeneity and suppressive immune microenvironment in glioblastoma Clinical trials of CAR T cells for glioblastoma (GBM) have identified several key challenges to therapeutic efficacy, including the inherently heterogeneous genomic landscape and immunosuppressive tumor microenvironment (TME) observed in GBM. Previous studies have shown that monovalent CAR T cells targeting EGFR variant III (EGFRvIII) reduce the target-positive tumor cell population, but tumor recurrence arises from target-negative tumor cells, highlighting the limitations of single-target approaches in heterogeneous tumors (O'Rourke et al., (2017). Sci Transl Med 9.10.1126 / scitranslmed.aaa0984). Regarding the highly immunosuppressive TME in GBM, transforming growth factor-β (TGFβ) was present in GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFβ is consistently highly expressed in both GBM tumor cell lines and patient tumor tissue (Figure 2A-2D).

[0417] A trivalent construct (CART-EGFR-IL13Rα2-dnTGFβ) was designed using two parallel scFv constructs independently targeting both IL13Rα2 and EGFRvIII, as well as a cleaved dominant-negative (dn) TGFβ receptor II (Figure 1, Figures 3A-3B). This trivalent construct was designed to explore potential additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFβ construct expanded the target tumor cell repertoire, blocked TGFβ signaling (Figure 3), and acted as a sink for free TGFβ in GBM TME to overcome the suppressive function of TGFβ.

[0418] The 3-module CAR T construct (i.e., the trivalent construct (CART-EGFR-IL13Rα2-dnTGFβ)) exhibited an enhanced proliferation response in vitro compared to the CART-EGFR-IL13Rα2 construct (Figure 7). In co-culture assays, this construct resulted in decreased PD-1 expression (Figure 5) and increased effector phenotype (Figures 9A-D) compared to the dicistronic CAR T construct, suggesting a lower proportion of exhausted T cells. The 3-module CAR T cells blocked the inhibitory pSmad2 / 3 signaling pathway, resulting in unimpeded activation (Figures 6A-6B) and an uninhibited proliferation response, although there was no significant enhancement of tumor-killing activity in short-term co-culture (Figures 4A-4B). In an immunodeficient mouse model, three-module CAR T cells safely and efficiently eradicated tumor cells compared to mice treated with dnTGFβ receptor II-deficient nicistorric CART-EGFR-IL13Rα2 cells, and the mice had a longer median survival time.

[0419] Overcoming adaptive changes in localized TMEs and addressing antigen heterogeneity is necessary to improve the clinical efficacy of CAR T-targeted strategies. This study demonstrated that the nicistronic CART construct works efficiently with cleaved TGFβ receptor II. Using dominant-negative TGFβRII in combination with CART-EGFR-IL13Rα2 offers several advantages, including CAR T cells suppressing immunosuppressive TGFβ signaling, exhibiting reduced PD-1 expression (Figure 5), demonstrating significant proliferative capacity in an in vitro chronic stimulation model (Figure 7), having no side effects in vivo, and exhibiting enhanced eradication of GBM tumors in vivo (Figures 10A-D, 11). In summary, the three-module CAR T construct described herein, TGFβRII CART-EGFR-IL13Rα2, addresses the clinical challenges of antigen heterogeneity and immunosuppressive TMEs in GBM.

[0420] Enumerated aspects The following embodiments are provided, but the numbering should not be interpreted as indicating a level of importance. Embodiment 1 provides the following: a) A first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain that binds human IL13Rα2, a transmembrane domain, and an intracellular domain, b) A second polynucleotide sequence encoding a second CAR, comprising a second antigen-binding domain that binds epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain, and c) The third polynucleotide sequence encoding the dominant-negative TGFβII receptor (DN-TGFβRII) Nucleic acids, including Embodiment 2 provides the following: The nucleic acid according to embodiment 1, wherein the first and / or second antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, Fab, single-strand variable fragment (scFv), or a single-domain antibody. Embodiment 3 provides the following: The first antigen-binding domain described above is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000061.tif4128 and HCDR3 includes the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and A nucleic acid according to any of the above embodiments, including the above. Embodiment 4 provides the following: A nucleic acid of any of the above embodiments, wherein the first antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 8, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 9. Embodiment 5 provides the following: A nucleic acid according to any of the above embodiments, wherein the first antigen-binding domain is a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11. Embodiment 6 provides the following: A nucleic acid in any of the above embodiments, wherein the first polynucleotide sequence encodes a CAR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or 24. Embodiment 7 provides the following: The aforementioned second antigen-binding domain, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), where HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence The heavy chain variable region includes TIFF2026509470000062.tif4128 and HCDR3 contains the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and A nucleic acid according to any of the above embodiments, including the above. Embodiment 8 provides the following: A nucleic acid of any of the above embodiments, wherein the second antigen-binding domain includes a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 32. Embodiment 9 provides the following: A nucleic acid according to any of the above embodiments, wherein the second antigen-binding domain is a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71. Embodiment 10 provides the following: A nucleic acid in any of the above embodiments, wherein the second polynucleotide sequence encodes a CAR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. Embodiment 11 provides the following: A nucleic acid according to any of the above embodiments, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. Embodiment 12 provides the following: The nucleic acid in any of the embodiments thereof, wherein the nucleic acid encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77 or 79. Embodiment 13 provides the following: The aforementioned transmembrane domain, Artificial hydrophobic sequences, as well as type I transmembrane proteins, alpha, beta, or zeta chains of T cell receptors, transmembrane domains of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154, or transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). A nucleic acid of any of the above embodiments, selected from the group consisting of the above. Embodiment 14 provides the following: A nucleic acid according to any of the above embodiments, wherein the transmembrane domain includes the transmembrane domain of CD8. Embodiment 15 provides the following: The nucleic acid according to embodiment 13, wherein the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha. Embodiment 16 provides the following: A nucleic acid according to any of the above embodiments, wherein the intracellular domain includes a co-stimulatory signaling domain and an intracellular...

Claims

1. A first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain that binds human IL13Rα2, a transmembrane domain, and an intracellular domain, A second polynucleotide sequence encoding a second CAR, which includes a second antigen-binding domain that binds epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain, and The third polynucleotide sequence encoding the dominant-negative TGFβ type II receptor (DN-TGFβRII) Nucleic acids, including

2. The nucleic acid according to claim 1, wherein the first and / or second antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-strand variable fragment (scFv), or a single-domain antibody.

3. The first antigen-binding domain described above is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence A heavy chain variable region containing HCDR3 containing the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and The nucleic acid according to any of the claims, including

4. The nucleic acid according to any one of the claims, wherein the first antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 8, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:

9.

5. The nucleic acid according to any one of the claims, wherein the first antigen-binding domain is a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.

6. The nucleic acid according to any one of the claims, wherein the first polynucleotide sequence encodes a CAR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43.

7. The second antigen-binding domain described above is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence A heavy chain variable region containing the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and The nucleic acid according to any of the claims, including

8. The nucleic acid according to any one of the claims, wherein the second antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:

32.

9. The nucleic acid according to any one of the claims, wherein the second antigen-binding domain is a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

10. The nucleic acid according to any one of the claims, wherein the second polynucleotide sequence encodes a CAR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75.

11. The nucleic acid according to any one of the claims, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

12. The nucleic acid according to any one of the claims, wherein the nucleic acid encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77 or 79.

13. The aforementioned transmembrane domain, Artificial hydrophobic sequences, as well as type I transmembrane proteins, alpha, beta, or zeta chains of T cell receptors, transmembrane domains of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154, or transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). A nucleic acid according to any of the claims, selected from the group consisting of the above.

14. The nucleic acid according to any one of the claims, wherein the transmembrane domain comprises the transmembrane domain of CD8.

15. The nucleic acid according to claim 9, wherein the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha.

16. The nucleic acid according to any one of the claims, wherein the intracellular domain comprises a co-stimulatory signaling domain and an intracellular signaling domain.

17. The intracellular domain, Co-stimulatory domains or variants thereof of proteins selected from the group consisting of TNFR superfamily proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or intracellular domains derived from killer immunoglobulin-like receptors (KIRs). The nucleic acid according to any of the claims, including

18. The nucleic acid according to any one of the claims, wherein the intracellular domain includes a 4-1BB co-stimulatory domain.

19. The nucleic acid according to any one of the claims, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d cytoplasmic signaling domains, or a variant thereof.

20. The nucleic acid according to any one of the claims, wherein the intracellular signaling domain includes the intracellular domain of CD3ζ.

21. A nucleic acid comprising a first polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain is A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence A heavy chain variable region containing HCDR3 containing the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and The nucleic acid, including the nucleic acid.

22. The nucleic acid according to claim 21, wherein the antigen-binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and / or a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

9.

23. The nucleic acid according to claim 21, wherein the antigen-binding domain is an scFv having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.

24. The nucleic acid according to claim 21, wherein the CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43.

25. The nucleic acid according to any one of claims 21 to 24, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

26. A first polynucleotide sequence encoding a CAR, comprising an antigen-binding domain that binds epidermal growth factor receptor (EGFR) or its isoform, a transmembrane domain, and an intracellular domain, and The second polynucleotide sequence encoding the dominant-negative TGFβ type II receptor (DN-TGFβRII) Nucleic acids containing these substances.

27. The nucleic acid according to claim 26, wherein the antigen-binding domain comprises a heavy chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31, and / or a light chain variable region having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:

32.

28. The nucleic acid according to claim 26, wherein the antigen-binding domain is an scFv having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71.

29. The nucleic acid according to claim 26, wherein the CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 35, 42, 43, or 75.

30. The nucleic acid according to claim 26, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

31. The nucleic acid according to claim 26, which encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81, 83, or 85.

32. A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR in the previous 1st year was, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1), and HCDR2 contains the amino acid sequence A heavy chain variable region containing HCDR3 containing the amino acid sequence DHRDAMDY (SEQ ID NO: 4), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7), and It includes an antigen-binding domain, The aforementioned second CAR is, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence A heavy chain variable region containing the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and It includes an antigen-binding domain, The aforementioned DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2. The nucleic acid.

33. A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The CAR in the previous 1st year was, SEQ ID NO: 44 or 54 and a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Includes, The aforementioned second CAR is, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, The nucleic acid.

34. A nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding IL13Rα2, a second polynucleotide sequence encoding a second CAR capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The first CAR comprises a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The second CAR comprises a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

70. The nucleic acid.

35. A nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding IL13Rα2, a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding epidermal growth factor receptor (EGFR) or its isoform, and a third polynucleotide sequence encoding DN-TGFβRII, The first polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO:

63. The aforementioned second polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

34. The nucleic acid.

36. The nucleic acid according to any one of claims 32 to 35, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

14.

37. The nucleic acid according to any one of the claims, wherein the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker.

38. The nucleic acid according to any one of the claims, wherein the second polynucleotide sequence and the third polynucleotide sequence are separated by a linker.

39. The nucleic acid according to any one of the claims, comprising the second polynucleotide sequence, a linker, and the first polynucleotide sequence from 5' to 3'.

40. The nucleic acid according to any one of the claims, comprising, from 5' to 3', the second polynucleotide sequence, a linker, the first polynucleotide sequence, a linker, and the third polynucleotide sequence.

41. A vector comprising the nucleic acid according to any of the above claims.

42. The vector according to claim 41, which is an expression vector.

43. A vector according to claim 41 or 42, selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

44. The vector according to any one of claims 41 to 43, further comprising an EF-1a promoter.

45. The vector according to any one of claims 41 to 44, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

46. A vector according to any one of claims 41 to 45, further comprising a rev response element (RRE).

47. The vector according to any one of claims 41 to 46, further comprising a cPPT sequence.

48. A self-inactivating vector, as described in any one of claims 41 to 47.

49. Modified immune cells or their progenitor cells comprising the nucleic acid according to any one of claims 1 to 40 or the vector according to any one of claims 41 to 48.

50. A first chimeric antigen receptor (CAR) containing a first antigen-binding domain capable of binding IL13Rα2, A second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding epidermal growth factor receptor (EGFR) or its isoform, Dominant-negative TGFβ type II receptor (DN-TGFβRII) and Modified immune cells or their progenitor cells, including those mentioned above.

51. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR in the previous 1st year was, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains and HCDR3 has an amino acid sequence A heavy chain variable region including, A light chain variable region containing three light chain complementarity-determining regions (LCDRs), wherein LCDR1 is an amino acid sequence The light chain variable region includes, where LCDR2 contains the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 17), and LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO: 7) or QHHYSAPWT (SEQ ID NO: 18). Includes, The aforementioned second CAR is, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence A heavy chain variable region containing the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and including, The modified immune cells or their progenitor cells.

52. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR in the previous 1st year was, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20. Includes, The aforementioned second CAR is, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

32. including, The modified immune cells or their progenitor cells.

53. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR comprises a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11. The second CAR comprises a single-stranded variable fragment (scFv) having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71. The modified immune cells or their progenitor cells.

54. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 42, or 43. The aforementioned second CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. The modified immune cells or their progenitor cells.

55. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The CAR in the previous 1st year was, SEQ ID NO: 44 or 54 and a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Includes, The aforementioned second CAR is, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, The modified immune cells or their progenitor cells.

56. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first CAR comprises an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The aforementioned second CAR includes an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

70. The modified immune cells or their progenitor cells.

57. A modified immune cell or its progenitor cell comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII), The first polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO:

63. The second polynucleotide sequence contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

34. The modified immune cells or their progenitor cells.

58. A modified immune cell or its progenitor cell according to any one of claims 51 to 57, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

14.

59. The aforementioned second CAR, Wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V , EGFR A289D , EGFR A289T , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and EGFR variant II A modified cell according to any one of claims 49 to 58, which can be conjugated with an EGFR isoform selected from the group consisting of the following, or any combination thereof.

60. The modified cell according to any one of claims 49 to 59, wherein the modified cell is a modified T cell.

61. The modified cell according to any one of claims 49 to 60, wherein the modified cell is an autologous cell.

62. The modified cells according to any one of claims 49 to 61, wherein the modified cells are autologous cells obtained from a human subject.

63. The modified cell according to any one of claims 49 to 62, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

64. A pharmaceutical composition comprising a therapeutically effective amount of modified cells according to any one of claims 49 to 63.

65. A method for treating a disease in a subject in need thereof, comprising the step of administering to the subject an effective amount of a modified cell according to any one of claims 49 to 63 or a pharmaceutical composition according to claim 64.

66. The method according to claim 65, wherein the disease is cancer.

67. The method according to claim 66, wherein the cancer is a glioma.

68. The method according to claim 65 or 66, wherein the cancer is an astrocytoma.

69. The method according to any one of claims 65 to 68, wherein the cancer is a high-grade astrocytoma.

70. The method according to any one of claims 65 to 68, wherein the cancer is glioblastoma.

71. A method for treating glioblastoma in subjects who require it, A first chimeric antigen receptor (CAR) containing a first antigen-binding domain capable of binding IL13Rα2, A second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding epidermal growth factor receptor (EGFR) or its isoform, Dominant-negative TGFβ type II receptor (DN-TGFβRII) and A step of administering an effective amount of modified T cells containing the above to a subject. The method, including the method described above.

72. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The CAR in the previous 1st year was, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), and HCDR2 contains the amino acid sequence It contains and HCDR3 has an amino acid sequence Heavy chain variable region including Includes, The aforementioned second CAR is, A heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 contains the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), and HCDR2 contains the amino acid sequence A heavy chain variable region containing the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27), A light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 contains the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 contains the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 contains the amino acid sequence VQYAQFPWT (SEQ ID NO: 30), and including, The aforementioned method.

73. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The CAR in the previous 1st year was, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 19, SEQ ID NO: 9 or 20 and the light chain variable region containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence Includes, The aforementioned second CAR is, A heavy chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and / or Light chain variable region containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

32. including, The aforementioned method.

74. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The first CAR comprises an scFv having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21 or SEQ ID NO:

22. The aforementioned second CAR includes an scFv containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33 or 71. The aforementioned method.

75. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The first CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 42 or SEQ ID NO:

43. The aforementioned second CAR contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35 or 75. The aforementioned method.

76. The method according to any one of claims 71 to 75, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

77. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The CAR in the previous 1st year was, SEQ ID NO: 44 or 54 and a heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, SEQ ID NO: 48 or 58 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Includes, The aforementioned second CAR is, A heavy chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74 and the light chain variable region encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. including, The aforementioned method.

78. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The first CAR comprises an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 69. The aforementioned second CAR includes an scFv encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

70. The aforementioned method.

79. A method for treating glioblastoma in subjects who require it, A process of administering an effective amount of modified T cells to a subject, comprising a first CAR capable of binding IL13Rα2, a second CAR capable of binding EGFR or its isoform, and a dominant-negative TGFβ type II receptor (DN-TGFβRII). Includes, The first CAR contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or SEQ ID NO: 53 or SEQ ID NO: 62 or SEQ ID NO:

63. The aforementioned second CAR contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

34. The aforementioned method.

80. The method according to any one of claims 77 to 79, wherein the DN-TGFβRII contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

14.

81. A method for treating glioblastoma in subjects who require it, The process of administering an effective amount of modified T cells to a subject, the modified T cells containing a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 or 78. The method, including the method described above.

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