Synthetic CAR for treating IL13Rα2-positive human and canine tumors

Abstract

To provide compositions and methods to treat IL13Rα2 positive tumors.SOLUTION: Provided are modified immune cells or precursors thereof (e.g., T cells) comprising chimeric antigen receptors (CARs) capable of binding human IL13Rα2. Also, provided are bispecific CARs, parallel CARs, tandem CARs, BiTEs, BiTE / CARs, and BiTE / BiTEs. Compositions and methods of treatment are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application takes priority to U.S. Provisional Patent Application No. 62 / 892,114, filed on 27 August 2019 under Section 119(e) of the U.S. Patent Act, 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 morbidity and mortality. The aggressive nature of the invasive growth of glioma cells within the central nervous system (CNS) makes total resection impossible. Despite the best available treatments, including surgical resection, radiotherapy, chemotherapy, and oncological treatment, the median survival is only 12–17 months for patients with GBM and 2–5 years for patients with grade III gliomas.

[0003] Redirected T-cell adoptive immunotherapy is a viable strategy for treating these malignancies. Long disease-free survival was achieved after treatment with CD19-targeted chimeric antigen receptor-modified autologous T (CAR) cells in patients with refractory chronic lymphocytic leukemia, and complete remission was achieved with this strategy 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 much more modest. Previously, humanized anti-EGFR variant III (EGFRvIII) CAR T cells (2173BBz) were used in a phase I clinical trial (NCT02209376) in 10 patients with relapsed GBM. Following CAR T cell infusion, there were clear changes in the tumor microenvironment, including a reduction in EGFRvIII target antigens associated with CAR T cell transport and functional activation in situ. However, this trial lacked the power to determine a clinical response (median overall survival was 251 days). A recent report described the use of repeated intratumoral and intrathecal infusions of redirected T cells expressing IL13 zetakin fused to a T cell signaling domain, a mutant IL13 cytokine, in one patient with relapsed multifocal GBM, which led to complete tumor regression over 7.5 months.

[0004] Interleukin-13 receptor α2 (IL13Rα2) is expressed in different human tumor types but is not found in normal human tissues except in adult testes (Figure 7B). 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 were also beneficial in pediatric glioma patients. While IL13 zetakin-redirected T cells bound to IL13Rα2 and induced a limited clinical response, these T cells also bound to IL13Rα1, which is expressed in some normal human tissues (Figure 7A), demonstrating harmful off-target effects.

[0005] The tumor microenvironment of malignant gliomas is immunosuppressive, as demonstrated after CAR T cell injection. Immune checkpoint receptors (e.g., PD-1, CTLA-4, TIM-3, and LAG-3) are a set of molecules that modulate T cell function by downregulating the stimulation of activated T cells in different spatiotemporal profiles. Checkpoint inhibitors are applied in cancer therapy to overcome T cell inhibition within the immunosuppressive tumor microenvironment and mobilize the T cell repertoire to target tumor cells. To date, most combination studies have used anti-PD-1 checkpoint blockade in conjunction with endogenous T cell responses to tumor antigens, with a select few reports relating to manipulated T cells.

[0006] There is a need in the art for compositions and methods for treating IL13Rα2-positive tumors. This invention addresses and responds to this need. [Overview of the project]

[0007] In one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to human IL13Rα2, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 7).

[0008] In certain embodiments, 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 SEQ ID NO:8. In certain embodiments, the antigen-binding domain includes 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. In certain embodiments, the antigen-binding domain includes a heavy 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:8; and 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.

[0009] In certain embodiments, the antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0010] In certain embodiments, the 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.

[0011] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0012] In certain embodiments, 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 SEQ ID NO:19. In certain embodiments, the antigen-binding domain includes 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:20. In certain embodiments, the antigen-binding domain includes a heavy 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:19; and 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:20.

[0013] In certain embodiments, the antigen-binding domain is selected from the group consisting of a full-length antibody or its antigen-binding fragment, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0014] In certain embodiments, the 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:21 or 22.

[0015] In certain embodiments, CAR can bind to IL13Rα2. In certain embodiments, CAR can bind to human IL13Rα2. In certain embodiments, CAR can bind to canine IL13Rα2. In certain embodiments, CAR can bind to both human IL13Rα2 and canine IL13Rα2.

[0016] In certain embodiments, the transmembrane domain is selected from the group consisting of artificial hydrophobic sequences, as well as transmembrane domains of type I transmembrane proteins, 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 transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). In certain embodiments, the transmembrane domain includes the transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha.

[0017] In certain embodiments, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domain includes a co-stimulatory domain of a protein selected from the group consisting of proteins within 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 variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). In certain embodiments, the intracellular domain includes a 4-1BB co-stimulatory domain. In certain embodiments, the intracellular signaling domain includes an intracellular domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor carrying an immunoreceptor-activated tyrosine motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or the cytoplasmic signaling domains of their variants. In certain embodiments, the intracellular signaling domain includes the intracellular domain of CD3ζ.

[0018] In another aspect, the present invention provides a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises 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; 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:9.

[0019] In another aspect, the present invention provides a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises 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:19; 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:20.

[0020] In another aspect, the present invention provides a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising 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.

[0021] In another aspect, the present invention provides nucleic acids comprising a polynucleotide sequence encoding any of the CARs intended herein.

[0022] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 7).

[0023] In certain embodiments, 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:57. In certain embodiments, the antigen-binding domain includes 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:61. In certain embodiments, 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:57; and 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:61.

[0024] In certain embodiments, the antigen-binding domain 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 or 133.

[0025] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0026] In certain embodiments, 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:67. In certain embodiments, the antigen-binding domain includes 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:71. In a particular 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:67; and 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:71.

[0027] In certain embodiments, the antigen-binding domain 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:134 or 135.

[0028] In certain embodiments, the transmembrane domain includes the transmembrane domain of CD8 alpha. In certain embodiments, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the co-stimulatory signaling domain includes the co-stimulatory domain of 4-1BB. In certain embodiments, the intracellular signaling domain includes the intracellular domain of CD3 ζ.

[0029] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. 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: 57; and 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: 61.

[0030] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, 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:67; and 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:71.

[0031] In another aspect, the present invention provides nucleic acids comprising a 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:75 or SEQ ID NO:76.

[0032] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the first CAR and the second CAR each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain, respectively.

[0033] In a particular embodiment, the antigen-binding domain of the first CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 7).

[0034] In a particular 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:57; and 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:61.

[0035] In a particular 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 or 133.

[0036] In a particular embodiment, the antigen-binding domain of the first CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0037] In a particular 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:67; and 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:71.

[0038] In a particular 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:134 or 135.

[0039] In a particular 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:65 or SEQ ID NO:66 or SEQ ID NO:75 or SEQ ID NO:76.

[0040] In a particular embodiment, the antigen-binding domain of the second CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 comprises the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0041] In certain embodiments, 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 SEQ ID NO:31. In certain embodiments, the antigen-binding domain of the second CAR includes 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. In a particular embodiment, the antigen-binding domain of the second CAR includes a heavy 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:31; and 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:32.

[0042] In certain embodiments, 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:33 or 141.

[0043] In a particular embodiment, the second polynucleotide sequence includes a 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:196.

[0044] In certain embodiments, the transmembrane domains of the first CAR and / or the second CAR are selected from the group consisting of artificial hydrophobic sequences, as well as transmembrane domains of type I transmembrane proteins, 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 transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). In certain embodiments, the transmembrane domains of the first CAR and / or the second CAR include a CD8 alpha transmembrane domain.

[0045] In certain embodiments, the intracellular domains of the first CAR and / or the second CAR include a co-stimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domains of the first CAR and / or the second CAR include a co-stimulatory domain of a protein selected from the group consisting of proteins within 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 variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). In certain embodiments, the intracellular domains of the first CAR and / or the second CAR include a 4-1BB co-stimulatory domain.

[0046] In certain embodiments, the intracellular signaling domain of the first CAR and / or the second CAR includes an intracellular domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor carrying an immunoreceptor-activated tyrosine motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or the cytoplasmic signaling domains of their variants. In certain embodiments, the intracellular signaling domain of the first CAR and / or the second CAR includes the intracellular domain of CD3ζ.

[0047] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), and LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID It contains NO:17) and includes a light chain variable region in which LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 comprises the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0048] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR 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: 57 or 67; and 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: 61 or 71. The second 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: 139 or 194; and 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: 140 or 195.

[0049] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. 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: 133, 134, 135, or 138; 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: 33 or 141.

[0050] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, 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:65 or SEQ ID NO:66 or SEQ ID NO:75 or SEQ ID NO:76; 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:35 or SEQ ID NO:196.

[0051] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding an immune checkpoint inhibitor.

[0052] In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-TIM-3 antibodies. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0053] In another aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform.

[0054] In a particular embodiment, the second polynucleotide sequence includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence encoding SEQ ID NO: 53 or 54.

[0055] In certain embodiments, BiTE can bind to wild-type EGFR (wtEGFR). In certain embodiments, BiTE can bind to EGFR variant III (EGFRvIII).

[0056] In certain embodiments, a first polynucleotide sequence and a second polynucleotide sequence are separated by a linker. In certain embodiments, the linker includes a nucleotide sequence encoding an intra-sequence ribosome entry site (IRES) or an autocleaved peptide. In certain embodiments, the autocleaved peptide is a 2A peptide. In certain embodiments, the 2A peptide is selected from the group consisting of porcine rhinitis virus-1 2A (P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A). In certain embodiments, the 2A peptide is T2A. In certain embodiments, the linker further includes a furin cleavage site.

[0057] In a particular embodiment, the nucleic acid comprises a first polynucleotide sequence, a linker, and a second polynucleotide sequence in the 5' to 3' direction. In a particular embodiment, the nucleic acid comprises a second polynucleotide sequence, a linker, and a first polynucleotide sequence in the 5' to 3' direction.

[0058] In a particular embodiment, the nucleic acid further comprises an inductive promoter, wherein the inductive promoter comprises a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NO: 161, 162, or 198.

[0059] In another aspect, the present invention provides a vector comprising any of the nucleic acids intended herein.

[0060] In certain embodiments, the vector is an expression vector. In certain embodiments, 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. In certain embodiments, the vector further comprises an EF-1a promoter. In certain embodiments, the vector further comprises a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the vector further comprises a rev response element (RRE). In certain embodiments, the vector further comprises a cPPT sequence. In certain embodiments, the vector is a self-inactivating vector.

[0061] In another aspect, the present invention provides modified immune cells or their progenitor cells comprising any of the CARs intended herein, any of the nucleic acids intended herein, or any of the vectors intended herein.

[0062] In another aspect, the present invention provides modified immune cells or their progenitor cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2. CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), and LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 16). It contains NO:17) and includes a light chain variable region in which LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18).

[0063] In another aspect, the present invention provides modified immune cells or their progenitor cells comprising a CAR capable of binding to IL13Rα2, wherein the CAR comprises a heavy 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: 8 or 19; and 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 or 20.

[0064] In another aspect, the present invention provides modified immune cells or their progenitor cells that include a CAR capable of binding to IL13Rα2, wherein the CAR includes 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.

[0065] In another aspect, the present invention provides modified immune cells or their progenitor cells containing a CAR capable of binding to IL13Rα2, 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:21 or 22.

[0066] In certain embodiments, CAR can bind to IL13Rα2. In certain embodiments, CAR can bind to human IL13Rα2.

[0067] In certain embodiments, the modified cells further contain an immune checkpoint inhibitor, in which case the modified cells secrete the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-TIM-3 antibody.

[0068] In certain embodiments, the modified cells further comprise an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, in which case the modified cells secrete the BiTE. In certain embodiments, the inducible BiTE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54. In certain embodiments, the BiTE is capable of binding to wild-type EGFR (wtEGFR). In certain embodiments, the BiTE is capable of binding to EGFR variant III (EGFRvIII).

[0069] In another aspect, the present invention provides modified immune cells or their progenitor cells comprising a first CAR comprising a first antigen-binding domain capable of binding to IL13Rα2; and a second CAR comprising a second antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoform.

[0070] In another aspect, the present invention provides modified immune cells or their progenitor cells comprising a first chimeric antigen receptor capable of binding to IL13Rα2, and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), and LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 16). It contains NO:17) and includes a light chain variable region in which LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 comprises the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0071] In another aspect, the present invention provides modified immune cells or their progenitor cells comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR comprises 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 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 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; 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.

[0072] In another aspect, the present invention provides modified immune cells or progenitor cells comprising a first chimeric antigen receptor capable of binding to IL13Rα2, and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein 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; and 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: 34.

[0073] In another aspect, the present invention provides a modified immune cell or a progenitor cell thereof, comprising a first chimeric antigen receptor capable of binding to IL13Rα2, and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, wherein the first CAR comprises 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; the second CAR comprises 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.

[0074] In certain embodiments, the modified cell further comprises an immune checkpoint inhibitor, wherein the modified cell secretes the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-TIM-3 antibody.

[0075] In certain embodiments, the CAR is capable of binding to human IL13Rα2.

[0076] In certain embodiments of the modified cell, 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 D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and is capable of binding to an EGFR isoform selected from the group consisting of EGFR variant II, or any combination thereof.

[0077] In certain embodiments, modified cells are modified immune cells. In certain embodiments, modified cells are modified T cells. In certain embodiments, modified cells are autologous cells. In certain embodiments, modified cells are autologous cells obtained from human subjects.

[0078] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the modified cells intended herein.

[0079] In another aspect, the present invention provides a method for treating a disease in a subject where such treatment is needed. The method comprises the step of administering an effective amount of any of the modified cells or any of the pharmaceutical compositions envisioned herein to the subject.

[0080] In certain aspects, the disease is cancer. In certain aspects, cancer is glioma. In certain aspects, cancer is astrocytoma. In certain aspects, cancer is high-grade astrocytoma. In certain aspects, cancer is glioblastoma.

[0081] In another aspect, 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 containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2 to the subject. CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), and LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID NO: 16). It contains NO:17) and includes a light chain variable region in which LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18).

[0082] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the 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.

[0083] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the 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 SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:22.

[0084] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, 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 or SEQ ID NO:24 or SEQ ID NO:55 or SEQ ID NO:56.

[0085] In certain embodiments, the method further includes a step of administering an immune checkpoint inhibitor, during which the modified cells secrete the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. In certain embodiments, the immune checkpoint inhibitor is administered co-administered with the modified T cells.

[0086] In a particular embodiment, the method further comprises the step of administering an inducible bispecific T cell engager (BiTE) capable of binding to the epidermal growth factor receptor (EGFR) or its isoform, wherein the modified cells secrete the BiTE.

[0087] In certain embodiments, inducible BiTE contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54. In certain embodiments, BiTE is capable of binding to wild-type EGFR (wtEGFR). In certain embodiments, BiTE is capable of binding to EGFR variant III (EGFRvIII). In certain embodiments, BiTE is administered co-administered with modified T cells.

[0088] In certain embodiments, the method further comprises the step of administering an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, and an immune checkpoint inhibitor, wherein the modified cells secrete the BiTE and the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is administered co-administered with the modified T cells.

[0089] In another aspect, the present invention provides a method for treating glioblastoma in a subject 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 to IL13Rα2; and a second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoform.

[0090] In another aspect, the present invention provides 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 chimeric antigen receptor capable of binding to IL13Rα2 and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform to a subject. The first CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), and LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6) or SASYRST (SEQ ID It contains NO:17) and includes a light chain variable region in which LCDR3 contains the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR is a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 comprises the amino acid sequence HGTNLDD (SEQ ID NO: 29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0091] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering an effective amount of modified T cells to a subject, comprising a first chimeric antigen receptor capable of binding to IL13Rα2 and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR comprises a heavy 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: 8 or 19; and 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 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; 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.

[0092] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering to a subject an effective amount of modified T cells comprising a first chimeric antigen receptor capable of binding to IL13Rα2 and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR comprises a single-stranded variable fragment (scFv) comprising 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) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34.

[0093] In another aspect, the present invention provides a method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering an effective amount of modified T cells to a subject comprising a first chimeric antigen receptor capable of binding to IL13Rα2 and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The first CAR comprises 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; the second CAR comprises 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.

[0094] In certain embodiments, the method further includes a step of administering an immune checkpoint inhibitor, during which the modified cells secrete the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. In certain embodiments, the immune checkpoint inhibitor is administered co-administered with the modified cells.

[0095] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a CAR, which includes a first antigen-binding domain, a second antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the first and second antigen-binding domains are separated by a linker. In certain embodiments, the linker comprises 5, 10, 15, or 20 amino acids. In certain embodiments, the first antigen-binding domain is capable of binding to IL13Rα2, and the second antigen-binding domain is capable of binding to epidermal growth factor receptor (EGFR) or its isoform. In certain embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 163, 165, 167, or 169. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NO: 164, 166, 168, or 170.

[0096] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a parallel CAR, wherein the parallel CAR comprises a first CAR and a second CAR, each comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, respectively, and the first CAR and the second CAR are separated by a cleavable linker. In a particular embodiment, the parallel CAR is encoded by a nucleotide sequence comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:171, and / or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:172.

[0097] In another aspect, the present invention provides nucleic acids comprising polynucleotide sequences encoding BiTE and CAR. In certain embodiments, BiTE comprises an antigen-binding domain capable of binding to EGFR or its isoform, and CAR comprises an antigen-binding domain capable of binding to IL13Rα2. In certain embodiments, BiTE comprises an antigen-binding domain capable of binding to IL13Rα2, and CAR comprises an antigen-binding domain capable of binding to EGFR or its isoform. In certain embodiments, the polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:176 or SEQ ID NO:178. In certain embodiments, the polynucleotide sequence is encoded by an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:175 or SEQ ID NO:177.

[0098] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a first BiTE and a second BiTE. In certain embodiments, the first BiTE and / or the second BiTE include an antigen-binding domain capable of binding to IL13Rα2 and / or an antigen-binding domain capable of binding to EGFR or its isoform. In certain embodiments, the polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:180. In certain embodiments, the 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:179. [Invention 1001] A chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to human IL13Rα2, a transmembrane domain, and an intracellular domain, The antigen-binding domain, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4); and 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). CAR, including. [Invention 1002] CAR 1001 of the present invention, comprising a heavy chain variable region in which the antigen-binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8. [Invention 1003] A CAR according to the present invention 1001 or 1002, comprising a light chain variable region in which the antigen-binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9. [Invention 1004] Any CAR of the present invention comprising: a heavy chain variable region having an antigen-binding domain that includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8; 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 SEQ ID NO:9. [Invention 1005] Any CAR of the present invention, wherein the 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. [Invention 1006] Any of the CARs of the present invention, wherein the 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. [Invention 1007] A chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain, The antigen-binding domain, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and 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). CAR, including. [Invention 1008] CAR of the present invention 1007, comprising a heavy chain variable region in which the antigen-binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:19. [Invention 1009] A CAR according to Invention 1007 or 1008, comprising a light chain variable region in which the antigen-binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:20. [Invention 1010] A CAR according to any one of the invention 1007 to 1009, comprising a heavy chain variable region in which the antigen-binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:19; and a light chain variable region in which an amino acid sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:20. [Invention 1011] A CAR according to any of the present invention 1007 to 1010, wherein the 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. [Invention 1012] A CAR according to any one of the inventions 1007 to 1011, wherein the 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: 21 or 22. [Invention 1013] A CAR according to any of invention 1001 to 1012, which is capable of binding with IL13Rα2. [Invention 1014] A CAR according to any of the invention 1001 to 1013, which is capable of binding to human IL13Rα2. [Invention 1015] A CAR according to any of invention 1001 to 1014, which is capable of binding to canine IL13Rα2. [Invention 1016] A CAR according to any of invention 1001 to 1015, which is capable of binding to human IL13Rα2 and canine IL13Rα2. [Invention 1017] A CAR according to any of the present invention 1001 to 1016, wherein 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). [Invention 1018] A CAR according to any of invention 1001 to 1017, wherein the transmembrane domain includes the transmembrane domain of CD8. [Invention 1019] The CAR of the present invention 1018, wherein the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha. [Invention 1020] A CAR according to any of invention 1001 to 1019, wherein the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. [Invention 1021] A CAR according to any of the Invention 1001 to 1020, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins within 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 variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). [Invention 1022] The CAR of the present invention 1021, wherein the intracellular domain includes a 4-1BB co-stimulatory domain. [Invention 1023] Any CAR according to invention 1020 to 1022, 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 the Fc receptor, a cytoplasmic receptor carrying an immunoreceptor-activated tyrosine motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or the cytoplasmic signaling domains of their variants. [Invention 1024] A CAR according to any one of the inventions 1020 to 1023, wherein the intracellular signaling domain includes the intracellular domain of CD3ζ. [Invention 1025] A chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8; and 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. CAR, including. [Invention 1026] A chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:19; and 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:20. CAR, including. [Invention 1027] A chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:55, or SEQ ID NO:56. [Invention 1028] A nucleic acid comprising a polynucleotide sequence encoding any of the CARs described in invention 1001 to 1028. [Invention 1029] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, including an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4); and 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). Nucleic acids, including [Invention 1030] The nucleic acid of the present invention 1029, 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:57. [Invention 1031] The nucleic acid of the present invention 1029 or 1030, wherein the antigen-binding domain comprises 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:61. [Invention 1032] A nucleic acid of any of the Invention 1029 to 1031, comprising: a heavy chain variable region whose antigen-binding domain 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: 57; and 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: 61. [Invention 1033] A nucleic acid according to any of the Invention 1029-1032, wherein the antigen-binding domain 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 or 133. [Invention 1034] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, including an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and 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). Nucleic acids, including [Invention 1035] The nucleic acid of the present invention 1034, 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:67. [Invention 1036] The nucleic acid of the present invention 1034 or 1035, wherein the antigen-binding domain comprises 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:71. [Invention 1037] A nucleic acid of any of the present inventions 1034 to 1036, comprising: a heavy chain variable region whose antigen-binding domain 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:67; and 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:71. [Invention 1038] A nucleic acid according to any of the Invention 1034-1037, wherein the antigen-binding domain 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: 134 or 135. [Invention 1039] A nucleic acid according to any of invention 1029 to 1038, wherein the transmembrane domain includes the transmembrane domain of CD8 alpha. [Invention 1040] A nucleic acid according to any of the Invention 1029 to 1039, wherein the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. [Invention 1041] The nucleic acid of the present invention 1040, wherein the co-stimulatory signaling domain includes a 4-1BB co-stimulatory domain. [Invention 1042] The nucleic acid of the present invention 1040 or 1041, wherein the intracellular signaling domain includes the intracellular domain of CD3ζ. [Invention 1043] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, including an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, Heavy chain variable regions encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:57; and 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:61. Nucleic acids, including [Invention 1044] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, including an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain, Heavy chain variable regions encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67; and 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:71. Nucleic acids, including [Invention 1045] Nucleic acids comprising a 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:75 or SEQ ID NO:76. [Invention 1046] A first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and A second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. A nucleic acid comprising a first CAR and a second CAR, each comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. [Invention 1047] The antigen-binding domain of the first CAR is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4); and 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). Nucleic acids of the present invention 1046, including the above. [Invention 1048] The nucleic acid of the present invention 1046 or 1047, wherein the antigen-binding domain of the first CAR 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: 57; and 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: 61. [Invention 1049] The nucleic acid of any of Invention 1046-1048, wherein 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 or 133. [Invention 1050] The antigen-binding domain of the first CAR is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and 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). Nucleic acids of the present invention 1046, including the above. [Invention 1051] The nucleic acid of the present invention 1050, wherein the antigen-binding domain of the first CAR 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:67; and 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:71. [Invention 1052] The nucleic acid of the present invention 1050 or 1051, wherein 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: 134 or 135. [Invention 1053] A nucleic acid according to any of Invention 1046 to 1053, wherein the first polynucleotide sequence 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:75 or SEQ ID NO:76. [Invention 1054] The antigen-binding domain of the second CAR is A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and 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). A nucleic acid comprising any of the inventions 1046 to 1053, including the above. [Invention 1055] The nucleic acid of the present invention 1054, wherein 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 SEQ ID NO:31. [Invention 1056] The nucleic acid of the present invention 1054 or 1055, wherein the antigen-binding domain of the second CAR includes 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:32. [Invention 1057] A nucleic acid of any of the Invention 1054 to 1056, wherein the antigen-binding domain of the second CAR comprises 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 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. [Invention 1058] The nucleic acid of any of Invention 1054 to 1057, wherein 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: 33 or 141. [Invention 1059] A nucleic acid according to any of Invention 1046 to 1058, wherein 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:35 or SEQ ID NO:196. [Invention 1060] The nucleic acids of any of Invention 1046 to 1059, wherein the transmembrane domains of the first CAR and / or the second CAR are selected from the group consisting of an artificial hydrophobic sequence and the transmembrane domains of type I transmembrane proteins, 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 transmembrane domains derived from killer immunoglobulin-like receptors (KIRs). [Invention 1061] A nucleic acid according to any of the Invention 1046-1060, wherein the transmembrane domain of the first CAR and / or the second CAR contains the transmembrane domain of CD8 alpha. [Invention 1062] The nucleic acid of any of the Invention 1046-1061, wherein the intracellular domains of the first CAR and / or the second CAR include a co-stimulatory signaling domain and an intracellular signaling domain. [Invention 1063] The nucleic acids of any of Invention 1046 to 1062 wherein the intracellular domain of the first CAR and / or the second CAR comprises a costimulatory domain of a protein selected from the group consisting of proteins within 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 variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). [Invention 1064] A nucleic acid according to any of the Invention 1046-1063, wherein the intracellular domains of the first CAR and / or the second CAR include a 4-1BB co-stimulatory domain. [Invention 1065] Any nucleic acid of the present invention 1046 to 1064, wherein the intracellular signaling domain of the first CAR and / or the second CAR comprises an intracellular domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, cytoplasmic tail of Fc receptor, cytoplasmic receptor carrying an immunoreceptor-activated tyrosine motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or the cytoplasmic signaling domains thereof. [Invention 1066] A nucleic acid according to any of the Invention 1046-1065, wherein the intracellular signaling domain of the first CAR and / or the second CAR includes the intracellular domain of CD3ζ. [Invention 1067] A first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and A second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. nucleic acids containing, The first car, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1) or SRNGMS (SEQ ID NO:12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO:3) or TVSSGGSYIYYADSVKG (SEQ ID NO:13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4) or QGTTALATRFFDV (SEQ ID NO:15); and 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) or KASQDVGTAVA (SEQ ID NO: 16), 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 comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and 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). Nucleic acids, including [Invention 1068] A first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and A second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. nucleic acids containing, The first car, Heavy chain variable regions encoded by polynucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57 or 67; and 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: 61 or 71. Includes; The second CAR, Heavy chain variable regions encoded by polynucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:139 or 194; and 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: 140 or 195. Nucleic acids, including [Invention 1069] A first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and A second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. nucleic acids containing, 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: 133, 134, 135, or 138; 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:33 or 141. Nucleic acid. [Invention 1070] A first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and A second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. nucleic acids containing, 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:65 or SEQ ID NO:66 or SEQ ID NO:75 or SEQ ID NO:76; The second polynucleotide sequence includes a 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:196. Nucleic acid. [Invention 1071] A nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding an immune checkpoint inhibitor. [Invention 1072] The nucleic acid of the present invention 1071, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [Invention 1073] The nucleic acid of the present invention 1071 or 1072, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. [Invention 1074] A nucleic acid according to any of the inventions 1071 to 1073, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody. [Invention 1075] A first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and A second polynucleotide sequence encoding an inducible bispecific T cell engager (BiTE) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms. Nucleic acids, including [Invention 1076] The nucleic acid of the present invention 1075, wherein the second polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence encoding SEQ ID NO: 53 or 54. [Invention 1077] The nucleic acid of the present invention 1075 or 1076, wherein BiTE is capable of binding to wild-type EGFR (wtEGFR). [Invention 1078] The nucleic acid of the present invention 1075 or 1076, wherein BiTE is capable of binding to EGFR variant III (EGFRvIII). [Invention 1079] A nucleic acid according to any of the inventions 1046 to 1078, wherein a first polynucleotide sequence and a second polynucleotide sequence are separated by a linker. [Invention 1080] The nucleic acid of the present invention 1079, wherein the linker comprises a nucleotide sequence encoding an intra-sequence ribosome entry site (IRES) or a self-cleaving peptide. [Invention 1081] The nucleic acid of the present invention 1080, wherein the self-cleaving peptide is a 2A peptide. [Invention 1082] The nucleic acid of the present invention 1081, wherein the 2A peptide is selected from the group consisting of porcine rhinitis virus-1 2A (P2A), Zhoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A). [Invention 1083] The nucleic acid of the present invention 1081 or 1082, wherein the 2A peptide is T2A. [Invention 1084] A nucleic acid according to any of the inventions 1079 to 1083, wherein the linker further comprises a furin cleavage site. [Invention 1085] A nucleic acid according to any of the Invention 1079 to 1084, comprising a first polynucleotide sequence, a linker, and a second polynucleotide sequence in the direction from 5' to 3'. [Invention 1086] A nucleic acid according to any of the Invention 1079 to 1084, comprising a second polynucleotide sequence, a linker, and a first polynucleotide sequence in the direction from 5' to 3'. [Invention 1087] A vector comprising any nucleic acid of the present invention 1029 to 1086. [Invention 1088] The expression vector, vector 1087 of the present invention. [Invention 1089] A vector according to the present invention 1087 or 1088, selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors. [Invention 1090] A vector according to any of invention 1087 to 1089, further comprising the EF-1a promoter. [Invention 1091] A vector according to any of the invention 1087 to 1090, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [Invention 1092] A vector according to any one of the invention 1087 to 1091, further comprising a rev response element (RRE). [Invention 1093] A vector further comprising a cPPT sequence, any of invention 1087 to 1092. [Invention 1094] A self-inactivating vector, any of the vectors described in invention 1087 to 1093. [Invention 1095] Modified immune cells or their progenitor cells comprising any CAR of Invention 1001 to 1027, any nucleic acid of Invention 1029 to 1086, or any vector of Invention 1087 to 1093. [Invention 1096] Modified immune cells or their progenitor cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1) or SRNGMS (SEQ ID NO:12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO:3) or TVSSGGSYIYYADSVKG (SEQ ID NO:13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4) or QGTTALATRFFDV (SEQ ID NO:15); and 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) or KASQDVGTAVA (SEQ ID NO: 16), 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). Modified immune cells or their progenitor cells, including those mentioned above. [Invention 1097] Modified immune cells or their progenitor cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8 or 19; and 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. Modified immune cells or their progenitor cells, including those mentioned above. [Invention 1098] Modified immune cells or their progenitor cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the 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. [Invention 1099] Modified immune cells or their progenitor cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21 or 22. [Invention 1100] A modified cell according to any of invention 1096 to 1099, wherein CAR is capable of binding to IL13Rα2. [Invention 1101] A modified cell according to any of the inventions 1096 to 1100, wherein the CAR is capable of binding to human IL13Rα2. [Invention 1102] A modified cell according to any of invention 1096 to 1101, further comprising an immune checkpoint inhibitor and secreting an immune checkpoint inhibitor. [Invention 1103] Modified cells of the present invention 1102, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [Invention 1104] Modified cells of the present invention 1102 or 1103, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. [Invention 1105] A modified cell according to any of the present invention 1096 to 1104, further comprising an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, and secreting BiTE. [Invention 1106] Modified cells of Invention 1105, wherein the inducible BiTE contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54. [Invention 1107] Modified cells of the present invention 1105 or 1106, wherein BiTE is capable of binding to wild-type EGFR (wtEGFR). [Invention 1108] Modified cells of the present invention 1105 or 1106, wherein BiTE is capable of binding to EGFR variant III (EGFRvIII). [Invention 1109] A first chimeric antigen receptor (CAR) comprising a first antigen-binding domain capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoform. Modified immune cells or their progenitor cells, including those mentioned above. [Invention 1110] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. Modified immune cells or their progenitor cells, The first car, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1) or SRNGMS (SEQ ID NO:12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO:3) or TVSSGGSYIYYADSVKG (SEQ ID NO:13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4) or QGTTALATRFFDV (SEQ ID NO:15); and 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) or KASQDVGTAVA (SEQ ID NO: 16), 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 comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and 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). Modified immune cells or their progenitor cells, including those mentioned above. [Invention 1111] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. Modified immune cells or their progenitor cells, The first car, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8 or 19; and 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 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. Modified immune cells or their progenitor cells, including those mentioned above. [Invention 1112] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. Modified immune cells or their progenitor cells, The first CAR comprises 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; 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:34. Modified immune cells or their progenitor cells. [Invention 1113] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. Modified immune cells or their progenitor cells, 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 24; 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. Modified immune cells or their progenitor cells. [Invention 1114] A modified cell according to any of invention 1109 to 1113, further comprising an immune checkpoint inhibitor and secreting an immune checkpoint inhibitor. [Invention 1115] Modified cells of the present invention 1114, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [Invention 1116] Modified cells of the present invention 1114 or 1115, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. [Invention 1115] A modified cell according to any of the invention 1096-1116, wherein the CAR is capable of binding to human IL13Rα2. [Invention 1116] 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 D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F Modified cells according to any of invention 1109 to 1115, which are capable of binding to an EGFR isoform selected from the group consisting of EGFR variant II, or any combination thereof. [Invention 1117] A modified immune cell, which is a modified cell according to any of the inventions 1096 to 1116. [Invention 1118] Modified T cells, which are modified cells according to any of the inventions 1096 to 1117. [Invention 1119] Autologous cells, modified cells according to any of the inventions 1096 to 1118. [Invention 1120] Autologous cells obtained from human subjects, modified cells according to any of the invention items 1096 to 1119. [Invention 1121] A pharmaceutical composition comprising a therapeutically effective amount of any modified cell according to invention 1096 to 1120. [Invention 1122] A method for treating a disease in a subject in need, comprising the step of administering an effective amount of any modified cells according to Invention 1096 to 1120, or a pharmaceutical composition according to Invention 1121, to the subject. [Invention 1123] The method of the present invention 1122, wherein the disease is cancer. [Invention 1124] The method of the present invention 1123, wherein the cancer is a glioma. [Invention 1125] The method of the present invention 1123 or 1124, wherein the cancer is an astrocytoma. [Invention 1126] A method according to any one of the present invention 1123 to 1125, wherein the cancer is a high-grade astrocytoma. [Invention 1127] The method according to any of the present invention 1123 to 1126, wherein the cancer is glioblastoma. [Invention 1128] A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1) or SRNGMS (SEQ ID NO:12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO:3) or TVSSGGSYIYYADSVKG (SEQ ID NO:13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4) or QGTTALATRFFDV (SEQ ID NO:15); and 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) or KASQDVGTAVA (SEQ ID NO: 16), 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). Methods that include... [Invention 1129] A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8 or 19; and 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. Methods that include... [Invention 1130] A method for treating glioblastoma in a subject requiring such treatment, comprising the step of administering to a subject an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the 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 SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:22. [Invention 1131] A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, 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 or SEQ ID NO:24 or SEQ ID NO:55 or SEQ ID NO:56. [Invention 1132] A method according to any one of the present invention 1128 to 1131, further comprising the step of administering an immune checkpoint inhibitor, wherein the modified cells secrete the immune checkpoint inhibitor. [Invention 1133] The method of the present invention 1132, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [Invention 1134] The method of the present invention 1132 or 1133, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIM-3 antibody. [Invention 1135] A method according to any one of the present invention 1132 to 1134, wherein an immune checkpoint inhibitor is administered simultaneously with modified T cells. [Invention 1136] Any method of the present invention 1128-1135, further comprising the step of administering an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the modified cells secrete the BiTE. [Invention 1137] The method of the present invention 1136, wherein the inducible BiTE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54. [Invention 1138] The method of the present invention 1136 or 1137, wherein BiTE is capable of binding to wild-type EGFR (wtEGFR). [Invention 1139] The method of the present invention 1136 or 1137, wherein BiTE is capable of binding with EGFR variant III (EGFRvIII). [Invention 1140] Any method 1136 to 1139 of the present invention, wherein BiTE is administered simultaneously with modified T cells. [Invention 1141] A method according to any of the present invention 1128-1131, further comprising the step of administering an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, and an immune checkpoint inhibitor, wherein the modified cells secrete the BiTE and the immune checkpoint inhibitor. [Invention 1142] The method of the present invention 1141, wherein BiTE and an immune checkpoint inhibitor are administered simultaneously with modified T cells. [Invention 1143] A first chimeric antigen receptor (CAR) containing a first antigen-binding domain capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) containing a second antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoform. A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing the specified substance to the target. [Invention 1144] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing the above, The first car, A heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1) or SRNGMS (SEQ ID NO:12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO:3) or TVSSGGSYIYYADSVKG (SEQ ID NO:13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4) or QGTTALATRFFDV (SEQ ID NO:15); and 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) or KASQDVGTAVA (SEQ ID NO: 16), 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 comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and 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). Methods that include... [Invention 1145] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing the above, The first car, Heavy chain variable regions containing amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8 or 19; and 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 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. Methods that include... [Invention 1146] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing the above, The first CAR comprises 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; 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:34. method. [Invention 1147] A first chimeric antigen receptor capable of binding to IL13Rα2, and A second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. A method for treating glioblastoma in a subject requiring treatment, comprising the step of administering an effective amount of modified T cells containing the above, 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 24; 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. method. [Invention 1148] A method according to any one of the present invention 1143 to 1147, further comprising the step of administering an immune checkpoint inhibitor, wherein the modified cells secrete the immune checkpoint inhibitor. [Invention 1149] The method of the present invention 1148, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [Invention 1150] The method of the present invention 1148 or 1149, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-TIM-3 antibodies. [Invention 1151] A method according to any of the present invention 1148 to 1150, wherein an immune checkpoint inhibitor is administered simultaneously with modified T cells. [Invention 1152] The nucleic acid of any of the Invention 1028 to 1086, further comprising an inductive promoter, wherein the inductive promoter comprises a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NO: 161, 162, or 198. [Invention 1153] A nucleic acid comprising a polynucleotide sequence encoding a CAR, which includes a first antigen-binding domain, a second antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the first antigen-binding domain and the second antigen-binding domain are separated by a linker. [Invention 1154] The nucleic acid of the present invention 1153, wherein the linker contains 5, 10, 15, or 20 amino acids. [Invention 1155] The nucleic acid of the present invention 1153, wherein the first antigen-binding domain is capable of binding to IL13Rα2, and the second antigen-binding domain is capable of binding to epidermal growth factor receptor (EGFR) or its isoform. [Invention 1156] The nucleic acid of the present invention 1155, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 163, 165, 167, or 169, and / or is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 164, 166, 168, or 170. [Invention 1157] A nucleic acid comprising a polynucleotide sequence encoding a parallel CAR, wherein the parallel CAR comprises a first CAR and a second CAR, each containing an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the first CAR and the second CAR are separated by a cleavable linker. [Invention 1158] The nucleic acid of the present invention 1157, wherein the parallel CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:171, and / or is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:172. [Invention 1159] A nucleic acid containing polynucleotide sequences encoding BiTE and CAR. [Invention 1160] The nucleic acid of the present invention 1159, wherein BiTE comprises an antigen-binding domain capable of binding to EGFR or its isoform, and CAR comprises an antigen-binding domain capable of binding to IL13Rα2. [Invention 1161] The nucleic acid of the present invention 1159, wherein BiTE comprises an antigen-binding domain capable of binding to IL13Rα2, and CAR comprises an antigen-binding domain capable of binding to EGFR or its isoform. [Invention 1162] The nucleic acid of the present invention 1159, wherein the polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:176 or SEQ ID NO:178. [Invention 1163] The nucleic acid of the present invention 1159, wherein the polynucleotide sequence is encoded by an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:175 or SEQ ID NO:177. [Invention 1164] A nucleic acid containing polynucleotide sequences encoding a first BiTE and a second BiTE. [Invention 1165] The nucleic acid of the present invention 1164, wherein the first BiTE and / or the second BiTE comprises an antigen-binding domain capable of binding to IL13Rα2 and / or an antigen-binding domain capable of binding to EGFR or its isoform. [Invention 1166] The nucleic acid of the present invention 1164, wherein the polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:180. [Invention 1167] The nucleic acid of the present invention 1164, wherein the 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:179. [Brief explanation of the drawing]

[0099] The aforementioned and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments made together with the accompanying drawings.

[0100] [Figure 1A] Figures 1A–1F illustrate humanized CAR T cells targeting IL13Rα2. Figure 1A shows flow cytometry detection of CAR expression in human T cells after electroporation of mRNA of mouse scFv and humanized scFv(07 and 08)-based CAR constructs using rabbit anti-mouse IgG or rabbit anti-human IgG antibodies. Figure 1B shows vector maps of the tested anti-IL13Rα2 CAR designs, based on the size of each component. Figure 1C illustrates CAR expression staining of humanized IL13Rα2 CAR transduced T cells used in co-culture experiments. Figure 1D shows expression analysis of IL13Rα1 and IL13Rα2 for human tumor cell lines (Sup-T1, Jurkat, A549, U87, U251, and D270). Figure 1E shows flow-based intracellular cytokine (IFNγ) staining of humanized IL13Rα2 CAR T cells co-cultured with human tumor cell lines in Figure 1D, with untransduced T cells (UTDs) as a control. Human CD8 staining was used to distinguish between CD4-positive and CD8-positive T cell subpopulations along the x-axis. Figure 1F shows a comparison of chromium release assay results from humanized IL13Rα2 CAR T cells co-cultured with tumor cell lines in Figure 1D at different effector / target (E:T) ratios (1:1, 3:1, 10:1, and 30:1) with untransduced T cells (UTDs) using a one-way ANOVA with a post-hoc Tukey test. **P<0.01, ***P<0.001, ****P<0.0001. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 1C] Refer to the explanation in Figure 1A. [Figure 1D] Refer to the explanation in Figure 1A. [Figure 1E] Refer to the explanation in Figure 1A. [Figure 1F] Refer to the explanation in Figure 1A. [Figure 2A]Figures 2A–2E illustrate the finding that IL13Rα2 CAR T cells regulate tumor growth in vivo. Figure 2A shows flow-based EGFRvIII and IL13Rα2 expression on the D270 tumor cell line, with a control antibody as the control. Figure 2B illustrates EGFRvIII-targeted (2173BBz) CAR T cells and IL13Rα2-targeted (Hu08BBz) CAR T cells co-cultured with the D270 tumor cell line. T cell stimulation was illustrated by staining with FITC conjugate anti-CD69 antibody, and after 24 or 48 hours of co-culture, the median fluorescence intensity (MFI) for CD4 CAR-positive T cells and CD8 CAR-positive T cells was quantified with non-transduced T cells as the control. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey test. Figure 2C illustrates the human T cells counted in the spleens (n=3) of D270-injected NSG mice 11 days after IV transfer of an equal number of non-transduced T cells, EGFRvIII-targeted (2173BBz) CAR T cells, or IL13Rα2-targeted (Hu08BBz) CAR T cells. Figure 2D illustrates the IV injection of 5 million CAR-positive EGFRvIII-targeted (2173BBz) CAR T cells or CAR-positive IL13Rα2-targeted (Hu07BBz and Hu08BBz) CAR T cells or the same number of non-transduced T cells 7 days after tumor implantation in an NSG mouse model (n=10 / group) subcutaneously implanted with D270. Tumor growth was assessed by measuring tumor volume (left panel) and bioluminescence imaging (center panel). Linear regression was used to test for significant differences between experimental groups. As predetermined IACUC-approved pathological condition endpoints, mice were defined as having their backs hunched, being unable to walk, or having a tumor reach 2 cm in any direction. Survival time was plotted based on time to the endpoint using Kaplan-Meier curves (Prism software). Statistical significance was determined using the log-rank test.Figure 2E illustrates that 800,000 IL13Rα2-targeted CAR-positive (Hu08BBz) CAR T cells or the same number of non-transduced T cells were delivered via IV injection to NSG mice (n=8 / group) orthotopically implanted with D270 tumors 8 days after tumor injection. Bioluminescence imaging was repeated every 3-4 days to assess tumor growth. Endpoints were predefined, and statistical significance was determined as shown in Figure 2D. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 2B] Refer to the explanation in Figure 2A. [Figure 2C] Refer to the explanation in Figure 2A. [Figure 2D] Refer to the explanation in Figure 2A. [Figure 2E] Refer to the explanation in Figure 2A. [Figure 3A]Figures 3A-3D illustrate the finding that checkpoint blockade selectively enhances the function of CAR T cells. Figure 3A shows that EGFRvIII (2173BBz) target-directed CAR T cells and IL13Rα2 (Hu08BBz) target-directed CAR T cells, as well as a control of untransduced T cells, were co-cultured with target-positive D270 tumor cell lines and target-negative A549 tumor cell lines. Checkpoint receptor expression on T cells was determined by flow cytometry and staining with a fluorescent dye conjugate anti-checkpoint receptor antibody; the median fluorescence intensity (MFI) for CD4 CAR-positive T cells and CD8 CAR-positive T cells was quantified after 24 or 48 hours of co-culture. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey test. Figure 3B shows that untransduced (UTD) human T cells were intravenously injected into a mouse model (n=5 / group) subcutaneously implanted with D270 7 days after tumor implantation. From day 6, PBS or 200 μg of the same volume of checkpoint blockade antibodies (anti-PD-1, anti-CTLA-4, and anti-TIM-3) were administered intraperitoneally every 4 days. Tumor size was measured and compared between the UTD+PBS group and the UTD+checkpoint blockade group. Figure 3C illustrates the same number of EGFRvIII-targeted (2173BBz) CAR T cells and IL13Rα2-targeted (Hu08BBz) CAR T cells injected as described in (Figure 3B) and combined with checkpoint blockade. Tumor volume in the checkpoint blockade combination therapy groups was compared to the CAR T cell control group combined with PBS (n=5 / group). Figure 3D illustrates the comparison of different checkpoint blockade combination therapies based on mouse tumor size in the EGFRvIII-targeted (2173BBz) CAR T cell group and the IL13Rα2-targeted (Hu08BBz) CAR T cell group. Survival curves were also compared for these two CAR T cell groups. Statistical significance of tumor growth between experimental groups was determined by linear regression, and a log-rank test was used to determine statistical significance of survival curves. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 3B] Refer to the explanation in Figure 3A. [Figure 3C] Refer to the explanation in Figure 3A. [Figure 3D] Refer to the explanation in Figure 3A. [Figure 4-1]Figures 4A–4E illustrate the finding that IL13Rα2 CAR T cells are selectively enhanced by in situ secreted anti-CTLA-4 checkpoint blockade. Figure 4A is a size-based vector map of each component of the design of minibody-secreted anti-IL13Rα2 CAR. The minibody is simplified as PD-1 target-directed scFv, CTLA-4 target-directed scFv, and TIM-3 target-directed scFv linked to a human IgG1 spacer and CH3 domain. The minibody was expressed together with the anti-IL13Rα2 CAR in the same open reading frame using a self-cleaved sequence (P2A). Figure 4B illustrates that CAR expression was detected not only on minibody-secreted IL13Rα2 target-directed CAR T cells but also on non-minibody-secreted IL13Rα2 target-directed CAR T cells. Figure 4C illustrates the collection and separate enrichment of supernatants from anti-PD-1 minibody-secreting IL13Rα2-targeted CAR T cells and anti-CTLA-4 minibody-secreting IL13Rα2-targeted CAR T cells. Standard direct ELISA was performed to assess the ability of anti-PD-1 and anti-CTLA-4 minibodies secreted by CAR T cells to bind recombinant hPD-1 and recombinant hCTLA-4. Statistical significance was calculated by unpaired t-tests. Figure 4D illustrates the co-culture of non-transduced T cells, IL13Rα2-targeted (Hu08BBz) CAR T cells, and minibody-secreting Hu08BBz CAR T cells with the D270 tumor cell line. Median fluorescence intensity (MFI) was quantified by BV605 conjugate anti-TIM-3 antibody staining of CD4 and CD8 subpopulations of CAR-positive T cells after 24 or 48 hours of co-culture. Statistical significance was calculated using one-way ANOVA with post-hoc Tukey test. Figure 4E illustrates the IV injection of 800,000 IL13Rα2 target-directed (Hu08BBz) CAR T cells and / or mini-body secretory Hu08BBz CAR T cells or the same number of non-transduced T cells 8 days (n=8) after subcutaneous implantation of D270. Tumor size was measured with calipers and compared between the groups. Statistical significance of tumor growth was determined by linear regression.ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 4-2] Refer to the explanation in Figure 4-1. [Figure 4-3] Refer to the explanation in Figure 4-1. [Figure 5A] Figures 5A–5D illustrate the finding that IL13Rα2 CAR T cells respond to canine tumors. Figure 5A shows the IL13Rα2 expression analysis of patient-derived glioma stem cell lines (5077, 5430, 4860, 5377, 5560, 4806, and 4892). Figure 5B illustrates the detection of CAR expression on IL13Rα2-targeted human CAR T cells (Hu07BBz and Hu08BBz) whose mRNA was electroporated. These CAR T cells were co-cultured with human IL13Rα2 protein and canine IL13Rα2 protein against bovine serum albumin (BSA) and then stained with intracellular cytokines (IFNγ). CD8 staining was used to distinguish between CD4-positive T cell groups and CD8-positive T cell groups on the x-axis. Figure 5C illustrates the detection of canine IL13Rα1 mRNA and IL13Rα2 mRNA expression in various canine tumor cell lines (Camac2, CLBL-1, GL-1, Cacal3, Cacal5, BW-KOSA, CS-KOSA, MC-KOSA, and SK-KOSA) by reverse transcription polymerase chain reaction (RT-PCR) using canine GAPDH as a control. After co-culturing IL13Rα2-targeted (Hu07BBz and Hu08BBz) human CAR T cells and untransduced T cells, which had their mRNA electroporated, with the aforementioned canine tumor cell lines, the percentage of cytokine (IFNγ, IL2, and TNFα)-positive T cells in the CD4-positive T cell subpopulation and CD8-positive T cell subpopulation was analyzed. Figure 5D illustrates the administration of 5 million MC-KOSA subcutaneously (n=5 / group) followed by 2 million Hu08BBz-transduced human CAR-positive T cells via intravenous injection 7 days later. Tumor size was measured using calipers and compared to a control group of the same amount of untransduced T cells. Statistical significance of tumor growth was determined by linear regression. ****P<0.0001. [Figure 5B]Refer to the explanation in Figure 5A. [Figure 5C] Refer to the explanation in Figure 5A. [Figure 5D] Refer to the explanation in Figure 5A. [Figure 6A] Figures 6A-6E illustrate the finding that canine IL13Rα2 CAR T cells control tumor growth in dogs. Figure 6A shows Hu08BBz canine CAR T cells, whose mRNA was electroporated, co-cultured with canine tumor cell lines (Camac2, CLBL-1, GL-1, Cacal3, Cacal5, BW-KOSA, CS-KOSA, MC-KOSA, SK-KPSA, and J3T). Canine IFNγ secretion was detected by ELISA and compared with stimulation by untransduced canine T cells. Figure 6B shows vector maps of the anti-IL13Rα2 human Hu08BBz CAR structure (Hu08HuBBz) and the canine Hu08BBz CAR structure (Hu08CaBBz). Figure 6C illustrates Hu07HuBBz, Hu08HuBBz, and Hu08CaBBz canine CAR T cells, whose mRNA was electroporated, co-cultured with CLBL1 and J3T tumor cell lines. Canine IFNγ secretion was detected by ELISA. The statistical significance of IFNγ secretion between Hu08HuBBz and Hu08CaBBz co-cultured with J3T glioma cells was determined using an unpaired t-test. Figure 6D illustrates J3T canine glioma cell lines orthotopically implanted in the brains of NSG mice. 12 million electroporated Hu08HuBBz, Hu08CaBBz, or untransduced canine T cells were intravenously injected into mouse models (n=4 / group) 7, 10, and 13 days after tumor implantation. Tumor growth was evaluated every 3-4 days by bioluminescence imaging. The statistical significance of tumor growth was determined by linear regression. Figure 6E illustrates the analysis of CAR expression and canine IFNγ secretion after co-culturing canine T cells used in the second injection on day 10 with a J3T tumor cell line. Canine CD4 staining was used to distinguish between canine CD4-positive and CD8-positive subpopulations along the x-axis. ns, not significant; **P<0.01, ****P<0.0001. [Figure 6B] Refer to the explanation in Figure 6A. [Figure 6C]Refer to the explanation in Figure 6A. [Figure 6D] Refer to the explanation in Figure 6A. [Figure 6E] Refer to the explanation in Figure 6A. [Figure 7A] Figures 7A–7C illustrate the IL13Rα1 and IL13Rα2 expression panels in normal human tissue and tumor tissue. Figures 7A–7B show the expression of IL13Rα1 and IL13Rα2 in normal human tissue, reported as mean TPM (parts per million transcripts), based on RNA-seq data from the Human Protein Atlas (HPA) (www.proteinatlas.org). Figure 7C shows the expression of IL13Rα2 in human tumors, described as the median expression based on cancer genome atlas (TCGA) data available on cBioPortal. [Figure 7B] Refer to the explanation in Figure 7A. [Figure 7C] Refer to the explanation in Figure 7A. [Figure 8A] Figures 8A–8D illustrate mouse scFv-based IL13Rα2 targeting CAR T cells. Figure 8A shows vector maps of tested mouse scFv-based anti-IL13Rα2 CAR designs. Figure 8B illustrates the expression of mouse scFv(07 and 08)-based IL13Rα2 targeting CAR constructs on electroporated human T cells. Figure 8C illustrates the expression analysis of IL13Rα1 and IL13Rα2 in human tumor cell lines (Sup-T1, Jurkat, U87, U251, and D270). Figure 8D illustrates flow-based intracellular cytokine (IFNγ) staining of mouse scFv-based IL13Rα2 CAR T cells (Mu07BBz and Mu08BBz) co-cultured with the human tumor cell lines in Figure 8C, with non-transduced T cells (UTDs) as a control. Human CD8 was stained, and the CD4-positive and CD8-positive subpopulations of T cells were identified along the x-axis. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 9A] Figures 9A-9C illustrate humanized IL13Rα2 target-directed CAR T cells co-cultured with human normal cell types. Figure 9A illustrates flow-based CAR expression staining of humanized IL13Rα2 CAR transduced T cells used in the co-culture experiment. Figure 9B illustrates flow cytometry of IL13Rα1 and IL13Rα2 expression analysis in human normal cells (CD34-positive myeloid cells, human pulmonary microvascular endothelial cells, human peripheral airway epithelial cells, human renal epithelial cells, human keratinocytes, human neural progenitor cells, human aortic smooth muscle cells, and human pulmonary artery smooth muscle cells). Figure 9C illustrates flow-based intracellular cytokine (IFNγ) staining of humanized IL13Rα2 CAR T cells co-cultured with human normal cells in Figure 9B, with non-transduced T cells (UTDs) as a control. Human CD3 and CD8 staining were used to distinguish between CD4-positive and CD8-positive subpopulations of T cells along the x-axis. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 10-1] Figures 10A–10E illustrate the stimulation and proliferation of IL13Rα2-targeted CAR T cells co-cultured in vitro. Figures 10A–10C illustrate flow-based intracellular cytokine (IFNγ, IL2, and TNFα) staining of mouse IL13Rα2 CAR T cells co-cultured with human tumor cell lines (Figure 10A), humanized IL13Rα2 CAR T cells co-cultured with human tumor cell lines (Figure 10B), and humanized IL13Rα2 CAR T cells co-cultured with human normal cells (Figure 10C). The percentage of cytokine-positive T cells in the CD4-positive and CD8-positive subpopulations is shown. Figure 10D illustrates flow-based EGFRvIII and IL13Rα2 expression in the D270 tumor cell line at days 0, 1, 2, 3, 5, and 7 in in vitro culture, using a control antibody as a control. Figure 10E illustrates the T cell proliferation assay determined by flow cytometry using CFSE staining on UTD T cells, 2173BBz CAR-positive T cells, and Hu08BBz CAR-positive T cells at days 3, 5, and 8, co-cultured with the D270 cell line, using the A549 cell line as a control. [Figure 10-2] Refer to the explanation in Figure 10-1. [Figure 11A] Figures 11A and 11B illustrate surface marker staining for CAR T cells co-cultured in vitro. Figure 11A shows a representative gating scheme illustrated using samples of UTD T cells, 2173BBz CAR T cells, and Hu08BBz CAR T cells co-cultured with the D270 cell line for 48 hours. Live CD45+ and CD3+ lymphocytes were gated, and the expression of T cell surface markers was analyzed and compared between CAR+ T cells and UTD T cells. Figure 11B illustrates the expression of CD69, PD-1, CTLA-4, and TIM-3 on CD4+ T cells and CD8+ T cells determined by flow cytometry after 24 or 48 hours of co-culture, using staining with corresponding antibodies conjugated with fluorescent dyes. Representative expression results for UTD T cells and CAR+ T cells co-cultured with the D270 cell line are illustrated. [Figure 11B] Refer to the explanation in Figure 11A. [Figure 12A]Figures 12A–12C illustrate the expression of checkpoint receptors and ligands involved in the in vivo activity of CAR T cells. Figure 12A illustrates flow-based detection of checkpoint receptors (PD-1, CTLA-4, and TIM-3) and their ligands (PD-L1, CD80, CD86, and galectin-9) in CD4-positive T cell subpopulations and CD8-positive T cell subpopulations at days 0, 3, 7, and 13 during in vitro expansion and proliferation of T cells using anti-CD3 and anti-CD28 beads. Figure 12B illustrates flow-based detection of checkpoint receptor ligand (PD-L1, CD80, CD86, and galectin-9) expression analysis in the D270 glioma cell line. Figure 12C illustrates the staining of human PD-1, CD69, CD4, and CD8 for human CD3+ T cells in ex vivo mouse spleens after injection of 2173BBz CAR T cells combined with anti-PD-1 checkpoint blockade in an NSG mouse model with subcutaneously implanted D270. Data are shown as the percentage of positive cells. Statistical significance was calculated by unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001. [Figure 12B] Refer to the explanation in Figure 12A. [Figure 12C] Refer to the explanation in Figure 12A. [Figure 13]Figures 13A-13C illustrate the in vitro analysis of mini-body secretory T cells (MiSTs) co-cultured with target cells. Figure 13A illustrates the co-culture of untransduced T cells, IL13Rα2 target-directed (Hu08BBz) CAR T cells, and mini-body secretory Hu08BBz CAR T cells (anti-PD1 and anti-CTLA4 MiSTs) with the D270 tumor cell line. After 24 or 48 hours of co-culture, the median fluorescence intensity (MFI) of the CD4 and CD8 subpopulations of CAR-positive T cells was quantified by staining with BV711 conjugate anti-PD1 antibody and PE conjugate anti-CTLA-4 antibody. Figure 13B illustrates the stimulation of IL13Rα2 (Hu08BBz) target-directed CAR T cells and mini-body secretory cells evaluated after co-culture with the D270 tumor cell line. The median fluorescence intensity (MFI) was quantified by FITC conjugate anti-CD69 antibody staining on CD4 and CD8 subpopulations of CAR-positive T cells after 24 or 48 hours of co-culture. Figure 13C illustrates the analysis of the percentage of cytokine (IFNγ, IL2, and TNFα) stain-positive T cells within the CD4-positive and CD8-positive T cell subpopulations for IL13Rα2-targeted (Hu08BBz) CAR T cells and minibody secretory cells after co-culture with D270-targeted tumor cell lines. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey test. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14A] Figures 14A-14B illustrate the amino acid sequences of IL13Rα2 and the canine osteosarcoma mouse model. Figure 14A shows a comparison of the amino acid sequences of human and canine IL13Rα2 using Geneious software. Figure 14B shows canine osteosarcoma tumor cell lines (BW-KOSA, CS-KOSA, MC-KOSA, and SK-KOSA) subcutaneously implanted in the right flank of NSG mice at different doses. Tumor growth in each group was evaluated by repeated bioluminescence imaging. [Figure 14B] Refer to the explanation in Figure 14A. [Figure 15]Figures 15A-15B illustrate the nucleotide sequences of the inducible promoters disclosed herein. Figure 15A: DNA sequence for an inducible promoter that can enhance expression after T cell activation. Partial repetition of this sequence can enhance T cell expression levels. This promoter can be used to modify T cells / CAR T cells to express designed RNA or amino acids. Figure 15B: Underlined sequence (SEQ ID NO: 198) that has been repeated to enhance activity. [Figure 16] Figures 16A and 16B illustrate the functional activity of the inducible promoter. Figure 16A is a schematic diagram of a construct containing the inducible promoter, including the TDTomato gene for fluorescence expression. Figure 16B shows the expression of TD-Tomato in Jurkat cells (T-cell tumor line) stimulated with PMA / ionomycin. When cells were stimulated with PMA / ionomycin, TD-Tomato expression was detected by flow cytometry, demonstrating promoter activation. [Figure 17] Tandem (top) and parallel (bottom) bispecific CARs are illustrated. The tandem bispecific CAR contains an IL13Rα2 antigen-binding domain (Hu08) linked to an EGFR antigen-binding domain (806). The linker in the tandem CAR can be 5, 10, 15, or 20 amino acids long (5AA / 10AA / 15AA / 20AA). The parallel CAR contains a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to EGFR. A self-cleaving sequence (P2A) links the anti-IL13Rα2 CAR and the anti-EGFR CAR in the same open reading frame. [Figure 18A] Figures 18A to 18E show the amino acid and nucleic acid sequences for a tandem CAR with a 5AA linker ((G4S); Figure 18A), a tandem CAR with a 10AA linker (2(G4S); Figure 18B), a tandem CAR with a 15AA linker (3(G4S); Figure 18C), a tandem CAR with a 20AA linker (4(G4S); Figure 18D), and a parallel CAR (Figure 18E). [Figure 18B]See the explanation in Figure 18A. [Figure 18C] See the explanation in Figure 18A. [Figure 18D] See the explanation in Figure 18A. [Figure 18E] See the explanation in Figure 18A. [Figure 19] This shows the quantification of CAR construct expression as determined by flow cytometry. T cells were transduced with Hu08BBz CAR, 806BBz CAR, Hu08 / 806_(G4S) bispecific CAR, Hu08 / 806_2(G4S) bispecific CAR, Hu08 / 806_3(G4S) bispecific CAR, Hu08 / 806_4(G4S) bispecific CAR, and Hu08BBz_P2A_806BBz parallel CAR. CAR expression was detected using either biotin-labeled protein L and streptavidin conjugate PE, or streptavidin conjugate PE alone. [Figure 20] The stimulation of T cells including Hu08BBz CAR and 806BBz CAR, Hu08 / 806 bispecific CAR, and Hu08BBz_P2A_806BBz parallel CAR is illustrated. Each CAR T cell population was co-cultured with 5077 glioma stem cell line overexpressing the target. CAR1 (Hu08BBz) and CAR2 (806BBz) were single CAR constructs, 5AA, 10AA, 15AA, and 20AA were tandem bispecific CAR constructs of varying lengths (Hu08 / 806_(G4S), Hu08 / 806_2(G4S), Hu08 / 806_3(G4S), Hu08 / 806_4(G4S)), and 2A was a parallel bispecific CAR construct (Hu08BBz_P2A_806BBz). T cell stimulation was visualized by staining with APC conjugate anti-CD69 antibody. After 24 hours of co-culture, the median fluorescence intensity (MFI) was quantified for CD4+ (Figure 20, top) CAR-positive T cells and CD8+ (Figure 20, bottom) CAR-positive T cells, using untransduced T cells as a control. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey test. *p<0.05, ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Figure 21-1] Figures 21A–21F illustrate flow-based intracellular cytokine staining [IFNγ (Figures 21A and 21D), IL2 (Figures 21B and 21E), and TNFα (Figures 21C and 21F)] of tandem bispecific CAR T cells and parallel CAR T cells co-cultured with 5077 glioma stem cell lines overexpressing the target, as shown in Figures 18–20. The percentage of cytokine-positive T cells in the CD4+ (Figures 21A–21C) T cell subpopulation and the CD8+ (Figures 21D–21F) T cell subpopulation was demonstrated. Post-hoc Tukey test of one-way ANOVA. **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Figure 21-2] See the explanation in Figure 21-1. [Figure 22A]Figures 22A-22D illustrate bioluminescence-based cytotoxicity assays performed to test the killing ability of 806 / Hu08 tandem bispecific CAR T cells when co-cultured with target 5077 cell lines that do not express EGFRvIII and IL13Rα2 (5077_Rα2-_vIII-), target 5077 cell lines that overexpress only IL13Rα2 (5077_Rα2+_vIII-), target 5077 cell lines that overexpress only EGFRvIII (5077_Rα2-_vIII+), or target 5077 cell lines that overexpress EGFRvIII and IL13Rα2 (5077_Rα2+_vIII+), as well as control untransduced T cells (UTDs). Figure 2A illustrates the bioluminescence-based cytotoxicity assay of Hu08 / 806_(G4S) bispecific CAR. The linker between the two scFvs is GGGGS (SEQ ID NO: 157). Data are presented as mean ± SEM. Figure 22B illustrates the bioluminescence-based cytotoxicity assay of the Hu08 / 806_2(G4S) bispecific CAR. The linker between the two scFvs is GGGGSx2 (SEQ ID NO: 181). Data are presented as mean ± SEM. Figure 22C illustrates the bioluminescence-based cytotoxicity assay of the Hu08 / 806_3(G4S) bispecific CAR. The linker between the two scFvs is GGGGSx3 (SEQ ID NO: 158). Data are presented as mean ± SEM. Figure 22D illustrates the bioluminescence-based cytotoxicity assay of the Hu08 / 806_4(G4S) bispecific CAR. The linker between the two scFvs is GGGGSx4 (SEQ ID NO: 160). The data is presented as mean ± SEM. [Figure 22B] Refer to the explanation in Figure 22A. [Figure 22C] Refer to the explanation in Figure 22A. [Figure 22D] Refer to the explanation in Figure 22A. [Figure 23]Figures 23A-23D illustrate the in vitro elimination of the parallel bispecific CAR construct (Hu08BBz_P2A_806BBz). Bioluminescence-based cytotoxicity assays were performed to test the toxicity of 806BBz / Hu08BBz (Hu08BBz_P2A_806BBz) parallel bispecific CAR T cells when co-cultured with target 5077 cell lines overexpressing only IL13Rα2 (5077_Rα2+_vIII-), target 5077 cell lines overexpressing only EGFRvIII (5077_Rα2-_vIII+), target 5077 cell lines overexpressing both EGFRvIII and IL13Rα2 (5077_Rα2+_vIII+), D270 glioma cell lines overexpressing both EGFRvIII and IL13Rα2 (D270_Rα2+_vIII+), and control untransduced T cells (UTD). Data are presented as mean ± SEM. [Figure 24] This figure illustrates how 806BBz / Hu08BBz (Hu08BBz_P2A_806BBz) parallel bispecific CAR T cells reduced tumor growth and improved animal survival. NSG mice (n=8 / group) subcutaneously implanted with D270 were intravenously injected with either 806BBz / Hu08BBz bispecific CAR T cells or the same number of untransduced T cells (UTDs). Tumor growth was assessed by measuring tumor volume (Figure 24, top). Linear regression was used to test for statistical significance between experimental groups. The endpoint was predefined as a predetermined IACUC-approved pathological condition endpoint, defined as the mouse hunching its back, being unable to walk, or the tumor reaching 2 cm in any direction. Survival time was plotted based on time to the endpoint using Kaplan-Meier curves (Figure 24, bottom, Prism software). Statistical significance was determined using the log-rank test. ****p<0.0001. Data are presented as mean ± SEM. [Figure 25]Figures 25A–25D illustrate T cell activation induced by the anti-IL13Rα2 / CD3 bispecific T cell engager (Hu07BiTE) in IL13Rα2-positive cells. Fresh medium (Figure 25A), as well as conditioned medium from untransduced (UTD) T cells (Figure 25B), Hu08BBz CAR-transduced T cells (Figure 25C), and Hu07BiTE-transduced T cells (Figure 25D), were collected and used for co-culture with cell line 5077 (top, IL13Rα2-) or cell line 4892 (bottom, IL13Rα2+). CD69 staining was used to demonstrate T cell activation. Human CD8 staining was used to differentiate between CD4-positive and CD8-positive subpopulations of T cells along the x-axis. [Figure 26] The in vitro binding of the anti-IL13Rα2 / CD3 (Hu08OKT3) bispecific T cell engager to IL13Rα2 is illustrated. 293 T cells were transfected with the plasmid pTRPE CFP (fluorescent gene) or pTRPE Hu08BiTE. The supernatant was collected after 2 days. Direct ELISA was performed to detect the binding of Hu08OKT3 BiTE to the recombinant protein IL13Rα2. [Figure 27] The in vitro binding of two anti-EGFR / CD3 (C225BiTE and 806BiTE) bispecific T cell engagers to EGFR is illustrated. T cells were transduced with pTRPE Hu08BBz, pTRPE C225BiTE, or pTRPE 806BiTE, and untransduced T cells (UTDs) and Hu8BBz CARs were used as controls. The supernatant was collected after 7 days. Direct ELISA was performed to detect the binding of BiTE to recombinant proteins, EGFR wild-type, or EGFRvIII. [Figure 28A]Figures 28A-28B illustrate the difference in the effects of two anti-EGFR / CD3 (C225BiTE and 806BiTE) bispecific T cell engagers on wild-type 5077 cells. Furthermore, the glioma stem cell line 5077 expresses low levels of EGFR but does not express IL13Rα2. Transgenic T cells of 806BiTE and C225BiTE were co-cultured with wild-type 5077 cells or 5077 cells overexpressing EGFRvIII, and a cell-killing assay (Figure 28A) and a cytokine secretion quantification assay (Figure 28B) were performed. Figure 28A illustrates that transgenic T cells of 806BiTE killed only EGFRvIII-overexpressing 5077 cells, while transgenic T cells of C225BiTE killed both wild-type and EGFRvIII-overexpressing 5077 cells. Figure 28B illustrates that when 806BiTE transduced T cells were co-cultured with EGFRvIII-overexpressing 5077 cells, 806BiTE induced the secretion of INFγ, IL-2, and TNF, whereas C225BiTE transduced T cells stimulated the secretion of INFγ, IL-2, and TNF both in the absence and in the presence of the EGFRvIII variant. No cytokine production was observed in the absence of target cells. [Figure 28B] Refer to the explanation in Figure 28A. [Figure 29] This paper illustrates T cell activation induced by the anti-IL13Rα2 / CD3(Hu08 / KT3-T2A-mCherry) and anti-EGFRvIII / CD3(80 / KT3-T2A-mCherry) bispecific T cell engagers in IL13Rα2-positive and EGFRvIII-positive cells. Supernatants of untransduced T cells (UTDs), 806BBz CAR T cells, 806BiTE T cells, Hu08BBz CAR T cells, and Hu08BiTE T cells were collected and used for co-culture with untransduced T cells and targeted overexpression 5077 GSC cell lines and D270 glioma cell lines. CD69 staining was used to demonstrate T cell activation. Human CD8 staining was used to differentiate between CD4-positive and CD8-positive T cell subpopulations along the x-axis. [Figure 30]Figures 30A-30D illustrate schematic diagrams of the bispecific constructs used in the BiTE / CAR experiment. Figure 30A shows a schematic diagram of a parallel bispecific polynucleotide sequence (806BBz / Hu08BBz bispecific construct) containing a first nucleotide sequence encoding Hu08BBz CAR, a second nucleotide sequence encoding 806BBz CAR, and a third nucleotide encoding a fluorescent marker. Figure 30B shows a schematic polynucleotide sequence (806BiTE / Hu08CAR bispecific construct) containing a first nucleotide sequence encoding an anti-EGFRvIII / CD3 bispecific T cell engager, a second nucleotide encoding Hu08CAR, and a third nucleotide encoding a fluorescent marker. Figure 30C shows a schematic polynucleotide sequence (Hu08BiTE / 806CAR bispecific construct) containing a first nucleotide sequence encoding an anti-IL13Rα2 / CD3 bispecific T cell engager, a second nucleotide encoding the 806CAR, and a third nucleotide encoding a fluorescent marker. Figure 30D shows a schematic polynucleotide sequence (806BiTE / Hu08BiTE bispecific construct) containing a first nucleotide sequence encoding an anti-EGFRvIII / CD3 bispecific T cell engager, a second nucleotide encoding the anti-IL13Rα2 / CD3 bispecific T cell engager, and a third nucleotide encoding a fluorescent marker. The self-cleaving sequence (P2A and / or T2A) ligates the CAR, bispecific T cell engager, and fluorescent marker into the same open reading frame. [Figure 31A] Figures 31A to 31D show the amino acid sequences and nucleic acid sequences for 806BBz / Hu08BBz (Figure 31A), described as SEQ ID No: 173 to 174; 806BiTE / Hu08BBz (Figure 31B), described as SEQ ID No: 175 to 176; Hu08BiTE / 806BBz (Figure 31C), described as SEQ ID No: 177 to 178; and 806BiTE / Hu08BiTE (Figure 31D), described as SEQ ID No: 179 to 180. [Figure 31B] Refer to the explanation in Figure 31A. [Figure 31C] Refer to the explanation in Figure 31A. [Figure 31D] Refer to the explanation in Figure 31A. [Figure 32] Figures 32A-32D illustrate bioluminescence-based cytotoxicity assays performed to test the killing ability of 806BiTE / Hu08BBz bispecific T cells when co-cultured with cell lines overexpressing the target (EGFRvIII / IL13Rα2) against non-transduced T cells (UTDs) as a control. Data are presented as mean ± SEM. Figure 32A shows the cytotoxic effect in the 5077 cell line (5077_Rα2-_vIII+) overexpressing only EGFRvIII. Figure 32B shows the cytotoxic effect in the 5077 cell line (5077_Rα2+_vIII-) overexpressing only IL13Rα2. Figure 32C shows the cytotoxic effect in the 5077 cell line (5077_Rα2+_vIII+) overexpressing both IL13Rα2 and EGFRvIII. Figure 32D shows the cytotoxic effect in the D270 cell line (D270_Rα2+_vIII+) overexpressing IL13Rα2 and EGFRvIII. [Figure 33]Figures 33A-33B show that 806BiTE / Hu08BBz bispecific T cells reduced tumor growth and improved animal survival. 806BiTE / Hu08BBz bispecific T cells or the same number of untransduced T cells (UTDs) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 33A shows the reduced tumor size in animals treated with 806BiTE / Hu08BBz bispecific T cells. Tumor growth was assessed by measuring tumor volume. Linear regression was used to test for significant differences between experimental groups. Endpoints were predefined as predetermined IACUC-approved pathological condition endpoints, such as the mouse hunching its back, being unable to walk, or the tumor reaching 2 cm in any direction. Figure 33B shows the improved survival in animals treated with 806BiTE / Hu08BBz bispecific T cells. Survival time was plotted based on time to the endpoint using Kaplan-Meier curves (Prism software). Statistical significance was determined using the log-rank test. ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Figure 34] Figures 34A–34D illustrate bioluminescence-based cytotoxicity assays performed to test the killing ability of Hu08BiTE / 806BBz bispecific T cells when co-cultured with 5077 cell lines and D270 glioma cell lines overexpressing the target (EGFRvIII / IL13Rα2), using untransduced T cells (UTDs) as a control. Data are presented as mean ± SEM. Figure 34A shows the cytotoxic effect in 5077 cell lines overexpressing only EGFRvIII (5077_Rα2-_vIII+). Figure 34B shows the cytotoxic effect in 5077 cell lines overexpressing only IL13Rα2 (5077_Rα2+_vIII-). Figure 34C shows the cytotoxic effect in 5077 cell lines overexpressing both IL13Rα2 and EGFRvIII (5077_Rα2+_vIII+). Figure 34D shows the cytotoxic effect in the D270 cell line (D270_Rα2+_vIII+) overexpressing IL13Rα2 and EGFRvIII. [Figure 35] Figures 35A–35B show that Hu08BiTE / 806BBz bispecific T cells reduced tumor growth and improved animal survival. Hu08BiTE / 806BBz bispecific T cells or the same number of untransduced T cells (UTDs) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 35A shows the reduced tumor size in animals treated with Hu08BiTE / 806BBz bispecific T cells. Tumor growth was assessed by measuring tumor volume. Linear regression was used to test for significant differences between experimental groups. Endpoints were predefined as predetermined IACUC-approved pathological condition endpoints, such as the mouse hunching its back, being unable to walk, or the tumor reaching 2 cm in any direction. Figure 35B shows the improved survival in animals treated with Hu08BiTE / 806BBz bispecific T cells. Survival time was plotted based on time to the endpoint using Kaplan-Meier curves (Prism software). Statistical significance was determined using the log-rank test. **p<0.01, ****p<0.0001. Data are presented as mean ± SEM. [Figure 36] Figures 36A-36D illustrate bioluminescence-based cytotoxicity assays performed to test the killing ability of 806BiTE / Hu08BiTE bispecific T cells when co-cultured with 5077 cell lines and D270 glioma cell lines overexpressing the target (EGFRvIII / IL13Rα2), using untransduced T cells (UTDs) as a control. Data are presented as mean ± SEM. Figure 36A shows the cytotoxic effect in 5077 cell lines overexpressing only EGFRvIII (5077_Rα2-_vIII+). Figure 36B shows the cytotoxic effect in 5077 cell lines overexpressing only IL13Rα2 (5077_Rα2+_vIII-). Figure 36C shows the cytotoxic effect in 5077 cell lines overexpressing both IL13Rα2 and EGFRvIII (5077_Rα2+_vIII+). Figure 36D shows the cytotoxic effect in the D270 cell line (D270_Rα2+_vIII+) overexpressing IL13Rα2 and EGFRvIII. [Figure 37] Figures 37A–37B show that 806BiTE / Hu08BiTE bispecific T cells reduced tumor growth and improved animal survival. 806BiTE / Hu08BiTE bispecific T cells or the same number of untransduced T cells (UTDs) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 37A shows the reduced tumor size in animals treated with 806BiTE / Hu08BiTE bispecific T cells. Tumor growth was assessed by measuring tumor volume. Linear regression was used to test for significant differences between experimental groups. Endpoints were predefined as predetermined IACUC-approved pathological condition endpoints, such as the mouse hunching its back, being unable to walk, or the tumor reaching 2 cm in any direction. Figure 37B shows the improved survival in animals treated with 806BiTE / Hu08BiTE bispecific T cells. Survival time was plotted based on time to the endpoint using Kaplan-Meier curves (Prism software). Statistical significance was determined using the log-rank test. ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Figure 38] To demonstrate the feasibility of intratumor injection, we illustrate the diffusion of the therapeutic agent into the contralateral ventricle following intraventricular injection into the other ventricle. 5 μL of trypan blue was injected into the right ventricle of the bregma tumor, 1–2 mm to the right, 0.3 mm anteriorly, and at a depth of 3.0 mm. The animals were euthanized within 15 minutes of injection, and the brain was examined for the diffusion of trypan blue into the contralateral ventricle. The blue staining observed in both ventricles indicates both that the therapeutic agent can be injected into the right ventricle and that diffusion of the therapeutic agent into the left contralateral ventricle can be achieved. [Modes for carrying out the invention]

[0101] Detailed explanation The present invention provides compositions and methods for modified immune cells or their progenitor cells (e.g., modified T cells) comprising a chimeric antigen receptor (CAR) capable of binding to human IL13Rα2. In some embodiments, the present invention provides compositions and methods for modified immune cells or their progenitor cells comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The provided compositions and methods are useful for treating cancers (e.g., glioma, high-grade astrocytoma, and glioblastoma).

[0102] Please understand that the methods described herein are not limited to, and the specific methods and / or experimental conditions disclosed herein may vary. Also, please understand that the terminology used herein is intended to describe only specific aspects and is not intended to be restrictive.

[0103] Furthermore, unless otherwise specified, the experiments described herein utilize conventional molecular, cellular, and immunological techniques within the scope of the skills of those skilled in the art. Such techniques are well known to skilled operators 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; 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).

[0104] A. Definition Unless otherwise specified, scientific and technical terms used herein have meanings that are widely understood by those skilled in the art. In the event of any potential semantic uncertainty, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise specifically required by the context, singular terms shall include plural terms, and plural terms shall include singular terms. Unless otherwise specified, the use of “or” shall mean “and / or.” The use of the term “including,” as well as other forms such as “includes” and “included,” is non-restrictive.

[0105] 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 widely used in the art. The methods and techniques provided herein are carried out according to conventional methods generally known in the art unless otherwise specified, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out according to the standards of manufacturers, as widely accomplished in the art or as described herein. The nomenclature and testing procedures and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as for patient treatment.

[0106] To make this disclosure easier to understand, selected terms are defined below.

[0107] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, “an element” means one element or more elements.

[0108] As used herein, "about" when referring to measurable values ​​such as quantity or duration of time includes a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, because such variation is reasonable for carrying out the disclosed method.

[0109] As used herein, “activation” refers to a state in which a T cell is sufficiently stimulated to induce detectable cell proliferation. Activation may also relate to induced cytokine production and detectable effector function. The term “activated T cell” specifically refers to a T cell undergoing cell division.

[0110] As used herein, “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0111] As used herein, the term “antigen” is defined as a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immune-qualified cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can act as an antigen.

[0112] Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will therefore encode an "antigen" as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded 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 more than one partial nucleotide sequence of a gene, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a "gene." It is readily apparent that antigens may be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0113] As used herein, the term “self” is intended to mean any material derived from the same individual that is later reintroduced into that individual.

[0114] A "costimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a non-limited costimulatory response by the T cell, such as proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0115] As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0116] A "disease" is a health condition in which an animal is unable to maintain homeostasis, and its health will continue to deteriorate unless the disease is treated. In contrast, a "disorder" in an animal is a health condition in which the animal can maintain homeostasis, but its health is less desirable than when it is not disordered. Leaving a disorder untreated does not necessarily lead to a further decline in the animal's health.

[0117] The term "downregulation," as used herein, refers to a reduction or loss of gene expression of one or more genes.

[0118] "Effective dose" or "therapeutic effective dose" is used interchangeably herein and refers 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 multitude of methods recognized in the art. Those skilled in the art will understand that the amount of composition administered herein is variable and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the treated mammal, the severity of the disease, and the specific compound being administered.

[0119] "Code" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA, and the biological properties resulting therefrom, which have either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and which act as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is usually a nucleotide sequence listed 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.

[0120] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0121] The term “epitope,” as used herein, is defined as a small chemical molecule on an antigen that can trigger an immune response and induce a B-cell response and / or a T-cell response. An antigen may have one or more epitopes. Most antigens have many epitopes; i.e., they are polyvalent. Generally, an epitope is approximately about 10 amino acids and / or sugars in size. Preferably, an epitope is about 4 to 18 amino acids, more preferably about 5 to 16 amino acids, even more most preferably 6 to 14 amino acids, more preferably about 7 to 12, and most preferably about 8 to 10 amino acids. Those skilled in the art will understand that, generally, the overall three-dimensional structure of a molecule is the primary criterion for antigen specificity rather than a specific linear sequence of the molecule, and thus this distinguishes one epitope from another. Based on this disclosure, the peptides used in the present invention may be epitopes.

[0122] As used herein, the term “exogenous” means any material introduced from or produced outside of an organism, cell, tissue, or system.

[0123] As used herein, the term “enlarged” means an increase in number, such as an increase in the number of T cells. In one embodiment, the number of T cells enlarged ex vivo is greater than the number initially present in the culture. In another embodiment, the number of T cells enlarged ex vivo is greater than the number of other cell types in the culture. As used herein, the term “ex vivo” means cells that have been removed from an organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0124] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0125] An "expression vector" is 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 can be supplied by a 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.

[0126] As used herein, “identity” refers to the identity of subunit sequences between two polymer molecules, and especially between two amino acid molecules, such as between two polypeptide molecules. Two amino acid sequences are identical if they have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of matching or identical positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer 10 amino acids long) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matching or identical, then the two amino acid sequences are 90% identical.

[0127] As used herein, the term “immune response” is defined as a cellular response to an antigen in which lymphocytes recognize an antigen molecule as a foreign substance, induce antibody formation, and / or activate lymphocytes to eliminate the antigen.

[0128] The term "immunosuppression" is used herein to refer to a reduction in the overall immune response.

[0129] "Isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from its natural coexisting material is "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0130] 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 into the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0131] 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 chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0132] 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. This term encompasses, in a subject, preferably a human, disrupting and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response.

[0133] In the context of this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0134] The term "oligonucleotide" typically refers to a short polynucleotide. It should be understood that while nucleotide sequences are represented by DNA sequences (i.e., A, T, C, G), RNA sequences (i.e., A, U, C, G) where "T" is replaced by "U" are also included.

[0135] Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences that are degenerate of each other and encode the same amino acid sequence. The phrase “nucleotide sequences encoding a protein or RNA” may also include introns, insofar as nucleotide sequences encoding proteins may, by type, contain introns.

[0136] Oral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0137] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and that they 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 any means available in the art, including 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.

[0138] 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 constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly known in the art as peptides, oligopeptides, and oligomers, and long chains, commonly known in the art as proteins, of which there are many types. Polypeptides include, among other things, 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.

[0139] As used herein with respect to antibodies, the term "specifically binding" means an antibody that recognizes a specific antigen but does not substantially 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 interspecies 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 different alleles of that antigen. 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 in relation to the interaction between an antibody, protein, or peptide and a second chemical species to mean that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If the antibody is specific to epitope "A", then the presence of molecules containing epitope A (or free, unlabeled A) in the reaction product containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0140] The term "stimulation" refers to a primary response induced by a stimulating molecule (e.g., the TCR / CD3 complex) binding to its homologous ligand, thereby mediating a signaling event, such as signaling mediated by the TCR / CD3 complex, in a non-limiting manner. Stimulation can mediate changes in the expression of certain molecules, such as the downregulation of TGF-beta and / or rearrangement of the cytoskeleton.

[0141] 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.

[0142] As used herein, "stimulating ligand" means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to a congenital binding partner on T cells (referred to herein as a "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, in particular, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0143] The term “subject” is intended to include organisms (e.g., mammals) capable of eliciting an immune response. As used herein, “subject” or “patient” may be human or a non-human mammal. Non-human mammals include livestock and pets, such as sheep, cattle, pigs, dogs, cats, and mice. Preferably, the subject is human.

[0144] A "target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, the target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0145] As used herein, the terms “T cell receptor” or “TCR” refer to a membrane protein complex involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. The TCR may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct 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.

[0146] As used herein, the term “therapeutic” means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of the disease state.

[0147] A "graft" refers to a biocompatible grid or donor tissue, organ, or cells that will be transplanted. Examples of grafts may 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 that will be administered to the host. For example, a graft can refer to nucleic acids or proteins.

[0148] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which exogenous nucleic acids are transferred to or introduced into host cells. “Transfected,” “transformed,” or “transduced” cells are those that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary target cells and their offspring.

[0149] When this term is used herein, “to treat” a disease means to reduce the frequency or severity of at least one sign or symptom of the disease or disorder that the subject is suffering from.

[0150] A "vector" is a composition of material containing isolated nucleic acids and usable to deliver isolated nucleic acids into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmidal and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0151] Scope: Throughout this disclosure, various aspects of the invention can be presented in the form of scope. It should be understood that the scope of scope is merely for convenience and simplification and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, scope descriptions should be considered to specifically disclose all possible sub-ranges and the individual numbers within those ranges. For example, a scope description such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0152] B. Chimeric antigen receptor The present invention provides a chimeric antigen receptor (CAR) capable of binding to IL13Rα2. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain. Compositions and methods for modified immune cells or their precursor cells, such as modified T cells, containing the CAR are also provided. Thus, in some embodiments, 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) containing the CAR, vector, or nucleic acid are also provided.

[0153] The target CAR of the present invention comprises an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain. The target CAR of the present invention may optionally include a hinge domain. Therefore, the target CAR of the present invention comprises an antigen-binding domain capable of binding to IL13Rα2, a hinge domain, a transmembrane domain, and an intracellular domain.

[0154] The antigen-binding domain may be functionally ligated to another domain of the CAR, such as a transmembrane domain or an intracellular domain (both as described elsewhere herein), for expression in cells. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is functionally ligated to a second nucleic acid sequence encoding the transmembrane domain, and further functionally ligated to a third nucleic acid sequence encoding the intracellular domain.

[0155] The antigen-binding domains described herein can 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 target CAR of the present invention may also include a hinge domain described herein. The target CAR of the present invention may also include a spacer domain described herein. In some embodiments, the antigen-binding domain, transmembrane domain, and intracellular domain are separated by a linker.

[0156] In certain embodiments, CAR can bind to human IL13Rα2. In certain embodiments, CAR can bind to canine IL13Rα2. In certain embodiments, CAR can bind to both canine IL13Rα2 and human IL13Rα2.

[0157] In one aspect, the present invention includes an isolated antigen receptor (CAR) comprising an antigen-binding domain capable of binding to human IL13Rα2, a transmembrane domain, and an intracellular domain. The antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 7).

[0158] In another aspect, the present invention comprises an isolated CAR comprising an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0159] The tolerable variations in CAR sequences will be known to those skilled in the art. For example, in some embodiments, the CAR 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 shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, or 18.

[0160] In another aspect, the present invention comprises an isolated CAR capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy 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:8; and 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.

[0161] In another aspect, the present invention comprises an isolated CAR capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises 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:19; 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:20.

[0162] In another aspect, the present invention includes an isolated CAR capable of binding to IL13Rα2, comprising 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.

[0163] In another aspect, the present invention includes an isolated CAR capable of binding to IL13Rα2, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:92 or SEQ ID NO:94 or SEQ ID NO:111 or SEQ ID NO:113.

[0164] In certain embodiments, CARs can bind to GBM stem cells.

[0165] antigen-binding domain The antigen-binding domain of a CAR is the extracellular region of the CAR for binding to specific target antigens, including proteins, carbohydrates, and glycolipids. In certain embodiments, the antigen-binding domain can bind to IL13Rα2. In certain embodiments, the antigen-binding domain can bind to human IL13Rα2. In certain embodiments, the antigen-binding domain can bind to canine IL13Rα2. In certain embodiments, the antigen-binding domain can bind to both human and canine IL13Rα2.

[0166] In certain embodiments, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO:8. In certain embodiments, the antigen-binding domain includes a light chain variable region containing the amino acid sequence of SEQ ID NO:9. In certain embodiments, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO:19. In certain embodiments, the antigen-binding domain includes a light chain variable region containing the amino acid sequence of SEQ ID NO:20.

[0167] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1, HCDR2 comprises the amino acid sequence of SEQ ID NO:2, and HCDR3 comprises the amino acid sequence of SEQ ID NO:4; and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:5, LCDR2 comprises the amino acid sequence of SEQ ID NO:6, and LCDR3 comprises the amino acid sequence of SEQ ID NO:7.

[0168] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1, HCDR2 comprises the amino acid sequence of SEQ ID NO:3, and HCDR3 comprises the amino acid sequence of SEQ ID NO:4; and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:5, LCDR2 comprises the amino acid sequence of SEQ ID NO:6, and LCDR3 comprises the amino acid sequence of SEQ ID NO:7.

[0169] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:12, HCDR2 comprises the amino acid sequence of SEQ ID NO:13, and HCDR3 comprises the amino acid sequence of SEQ ID NO:14; and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:16, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:18.

[0170] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:12, HCDR2 comprises the amino acid sequence of SEQ ID NO:13, and HCDR3 comprises the amino acid sequence of SEQ ID NO:15; and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:16, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:18.

[0171] In certain embodiments, 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 certain embodiments, the antigen-binding domain includes an scFv capable of binding to IL13Rα2. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO:10. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO:11. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO:21. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO:22.

[0172] In certain embodiments, 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 certain embodiments, the antigen-binding domain includes an scFv capable of binding to IL13Rα2. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 125. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 127. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 129. In certain embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 131.

[0173] The tolerable 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 shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0174] The tolerable 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 shown in SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0175] The tolerable 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 shown in SEQ ID NO: 2, 77, 79, 82, 84, 86, 88, 90, 127, or 129.

[0176] The tolerable 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 shown in SEQ ID NO: 96, 98, 99, 100, 103, 105, 107, 109, 129, or 131.

[0177] The antigen-binding domain may contain any domain that binds to an antigen, and may include, but is not limited to, 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 may include 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.

[0178] 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.

[0179] As used herein, the terms “single-chain variable fragment” or “scFv” refer to a fusion protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and light chain (VL) are either directly linked or linked by a linker-coding peptide, which 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., the IL13Rα2-binding domain) includes an scFv having a VH-linker-VL configuration from N-terminus to C-terminus. In some embodiments, the antigen-binding domain includes an scFv having a VL-linker-VH configuration from N-terminus to C-terminus. Those skilled in the art will be able to select a configuration suitable for use in the present invention.

[0180] Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. Linkers can link 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. Glycine-serine (GS) linkers, e.g., (GS) n (GSGGS) n (SEQ ID NO:148), (GGGS) n (SEQ ID NO:149), and (GGGGS) nVarious linker sequences are known in the art, including, but not limited to, (SEQ ID NO:150) (where n is an integer of at least 1). Exemplary linker sequences include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO:151), GGSGG (SEQ ID NO:152), GSGSG (SEQ ID NO:153), GSGGG (SEQ ID NO:154), GGGSG (SEQ ID NO:155), GSSSG (SEQ ID NO:156), GGGGS (SEQ ID NO:157), GGGGSGGGGSGGGGS (SEQ ID NO:158), etc. Those skilled in the art will be able to select a linker sequence suitable for use in the present invention. In one embodiment, the antigen-binding domain of the present invention includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH and VL are nucleic acid sequences It is separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 158), which can be encoded by TIFF0007933412000001.tif12148.

[0181] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains its intrinsic immunoglobulin specificity. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patents Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publications Nos. 20050196754 and 20050196754. Inhibitory antagonist scFvs have 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) 2 (See 10:31-40). Agonist scFvs with stimulating activity have been 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).

[0182] 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 yields 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).

[0183] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of the entire IgG antibody, in which this fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains linked by disulfide bonds: a portion of the heavy chain and a light (L) chain for binding to the antigen, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0184] In some embodiments, the antigen-binding domain may originate from the same species in which the CAR will ultimately be 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 from the one in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may contain a mouse antibody or a fragment thereof.

[0185] 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 target cells. 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 may also bind to separate epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, these binding domains may be arranged in tandem and separated by linker peptides. 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 oligopeptide linker or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0186] 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 in question is a region capable of penetrating the plasma membrane of a cell (e.g., an immune cell or its progenitor cell). The transmembrane domain is intended for insertion into the cell membrane, for example, the eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0187] In some embodiments, the transmembrane domain is naturally associated with one or more domains in the CAR. In some embodiments, the transmembrane domain can be modified by selection or one or more amino acid substitutions to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0188] 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 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 of specific use 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, and TLR9 (i.e., including at least these transmembrane regions), or transmembrane domains derived from killer cell immunoglobulin-like domains (KIRs). In one embodiment, the transmembrane domain includes the transmembrane domain of CD8. In one embodiment, the transmembrane domain of CD8 includes the transmembrane domain of CD8α.

[0189] In some embodiments, the transmembrane domain may be synthetic, in which case it would primarily consist of hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine would be found at each terminus of the synthetic transmembrane domain.

[0190] The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the intracellular domains described herein, or any other domains described herein that may be included in the target CAR.

[0191] In some embodiments, the transmembrane domain further comprises a hinge region. The CARs of the present invention may also include a hinge region. The hinge region of a 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 include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that may be as small as three glycine (Gly) molecules, and the CH1 and CH3 domains of IgG (such as human IgG4).

[0192] In some embodiments, the CARs covered by this disclosure include a hinge region that connects an antigen-binding domain to a transmembrane domain and then to an intracellular domain. The hinge region is preferably capable of assisting the antigen-binding domain in recognizing and binding to a target antigen on a target cell (see, for example, Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a mobile domain, which in turn allows 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., above). The mobility of the hinge region allows it to assume many different conformations.

[0193] 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).

[0194] 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.

[0195] An appropriate hinge region can be easily selected and can be any of the following: 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including several appropriate lengths, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. An appropriate hinge region can have a length of 20 amino acids (e.g., 30, 40, 50, 60, or more amino acids).

[0196] For example, the hinge region is made of glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n (GSGGS) n (SEQ ID NO:148) and (GGGS) nThe material includes (SEQ ID NO:149), where n is at least an integer of 1, glycine-alanine polymers, alanine-serine polymers, and other mobile linkers known in the art. Glycine polymers and glycine-serine polymers can be used; both Gly and Ser are relatively structurally indeterminate and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine accesses significantly more φ-ψ 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). Exemplary hinge regions are non-limiting. It can include amino acid sequences such as TIFF0007933412000002.tif13164.

[0197] 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 has the following amino acid sequence: It can include one of the following: TIFF0007933412000003.tif49159, etc.

[0198] The hinge region may contain amino acid sequences of human IgG1, IgG2, IgG3, or IgG4 hinge regions. 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 in the human IgG1 hinge may be substituted with Tyr, thereby the hinge region containing the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 85); see, for example, Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region may contain amino acid sequences or variants thereof derived from human CD8.

[0199] intracellular domain The CARs of the present invention also include an intracellular domain. In some embodiments, 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, directing the cell (e.g., an immune cell) to perform its specific function, e.g., damage and / or destruction of target cells.

[0200] 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 cooperate 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.

[0201] Examples of intracellular domains include, but are not limited to, the zeta chain of the T cell receptor complex or any of its homologs, e.g., 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, an immunoreceptor tyrosine activation motif (ITAM)-containing cytoplasmic receptor, and combinations thereof.

[0202] In certain embodiments, the intracellular domain of a CAR includes any portion of one or more costimulatory molecules, e.g., at least one signaling domain from 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 any combination thereof. The intracellular domain includes a costimulatory domain of a protein selected from the group consisting of proteins within 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 variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). In certain embodiments, the intracellular domain includes a 4-1BB co-stimulatory domain.

[0203] 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 Rlla, 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, and 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 This includes, but is 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, fragments or domains derived from one or more molecules or receptors.

[0204] Additional examples of intracellular domains include, but are not limited to, intracellular signaling domains of several other diverse immune signaling receptors, but are not limited to, first, second, and third-generation T cell signaling proteins, including CD3, B7 family costimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, for example, Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), e.g., NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z signaling domains.

[0205] In certain embodiments, 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 carrying an immunoreceptor-activated tyrosine motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or the cytoplasmic signaling domains of their variants. In certain embodiments, the intracellular domain includes the intracellular domain of CD3ζ.

[0206] An intracellular domain suitable for use in the CAR of the present invention comprises any desired signaling domain that provides a distinct and detectable signal in response to the activation of the CAR (i.e., activation by an antigen and a dimerizing agent) (e.g., increased production of one or more cytokines by the cell; alteration of transcription of a target gene; alteration of protein activity; alteration of cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of cellular signaling responses, etc.). In some embodiments, the intracellular domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular domain comprises a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular domain is diffused into the cytoplasm instead of being covalently bound to the membrane-bound CAR.

[0207] The intracellular domain suitable for use in the target CAR of the present invention comprises an intracellular signaling polypeptide containing an immune receptor tyrosine activation motif (ITAM). In some embodiments, the ITAM motif is repeated twice within the intracellular domain, where the first and second examples of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular domain of the target CAR contains three ITAM motifs.

[0208] In some embodiments, the intracellular domain includes, non-limitingly, the signaling domain of a human immunoglobulin receptor containing an immune receptor tyrosine activation motif (ITAM), such as Fc-gamma-RI, Fc-gamma-RIIA, Fc-gamma-RIIC, Fc-gamma-RIIIIA, or FcRL5 (see, for example, Gillis et al., Front. Immunol. (2014) 5:254).

[0209] A suitable intracellular domain can be an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide. For example, a suitable intracellular domain can 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).

[0210] 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-activated receptor-related protein; killer-activated receptor-related protein, etc.). In another 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 immunoglobulin E receptor, high-affinity gamma chain, 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 another 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 yet another 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 another 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.

[0211] Typically, the entire intracellular domain can be used, but in many cases, it is not necessary to use the entire chain. Within the scope to which cleaved portions of the intracellular domain are used, such cleaved portions can be used in place of the intact chain, as long as they transmit the effector functional signal. The intracellular domain contains any cleaved portions of the intracellular domain sufficient to transmit the effector functional signal.

[0212] The intracellular domains described herein may 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.

[0213] (Table 1) Sequence used in the present invention TIFF0007933412000004.tif70157TIFF0007933412000005.tif245157TIFF0007933412000006.tif244157TIFF0007933412000007.tif243157TIFF0007933412000008.tif247157TIFF0007933412000009.tif247157TIFF0007933412000010.tif243157TIFF0007933412000011.tif244157TIFF0007933412000012.tif243157TIFF0007933412000013.tif243157TIFF0007933412000014.tif243157TIFF0007933412000015.tif243157TIFF0007933412000016.tif244157TIFF0007933412000017.tif243157TIFF0007933412000018.tif242157TIFF0007933412000019.tif245157TIFF0007933412000020.tif247157TIFF0007933412000021.tif243157TIFF0007933412000022.tif243157TIFF0007933412000023.tif247157TIFF0007933412000024.tif243157TIFF0007933412000025.tif243157TIFF0007933412000026.tif243157TIFF0007933412000027.tif150157

[0214] C. Tandem bispecific CAR and parallel bispecific CAR Tandem CARs, cells containing tandem CARs (e.g., T cells), amino acid sequences containing tandem CARs, and nucleic acids encoding tandem CARs are also provided herein. A tandem CAR comprises two antigen-binding domains separated by a linker, which are linked to a transmembrane domain and an intracellular domain (e.g., 4-1BB and / or CD3ζ) (Figure 17). In one aspect, a tandem 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) (Figure 17). The first and second antigen-binding domains can bind to two different antigens. For example, an exemplary tandem CAR comprises a first antigen-binding domain containing an scFv capable of binding to IL13Rα2, and a second antigen-binding domain containing an scFv capable of binding to EGFR.

[0215] The linker within the tandem CAR that connects the first antigen-binding domain and the second antigen-binding domain can be of various sizes, e.g., any number of amino acid lengths (Figures 18A-18D). For example, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths. In a particular embodiment, the tandem CAR includes a linker that is 5 amino acid lengths long. In a particular embodiment, the tandem CAR includes the amino acid sequence of SEQ ID NO: 163 and may be encoded by the nucleotide sequence of SEQ ID NO: 164. In a particular embodiment, the tandem CAR includes a linker that is 10 amino acid lengths long. In a particular embodiment, the tandem CAR includes the amino acid sequence of SEQ ID NO: 165 and may be encoded by the nucleotide sequence of SEQ ID NO: 166. In certain embodiments, the tandem CAR includes a linker that is 15 amino acids long. In certain embodiments, the tandem CAR includes the amino acid sequence of SEQ ID NO: 167 and may be encoded by the nucleotide sequence of SEQ ID NO: 168.

[0216] 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 separate 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 cleavable linker (e.g., a 2A linker) and a second transmembrane domain and a second intracellular domain. When the nucleic acid is expressed intracellularly, the linker (e.g., a 2A linker) is cleaved, and two separate CARs are expressed on the cell surface. In certain embodiments, the parallel CAR comprises a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to EGFR. In certain embodiments, the parallel CAR comprises an amino acid sequence with SEQ ID NO:171 and may be encoded by a nucleotide sequence with SEQ ID NO:172.

[0217] D. The combinations of BiTE, BiTE / BiTE, and BiTE / CAR Bispecific T cell engagers (BiTEs) and BiTE / CAR combinations are provided herein. A BiTE comprises a first antigen-binding domain (e.g., a first scFv) and a second antigen-binding domain (e.g., a second scFv), wherein the first scFv is capable of binding to an antigen on a target cell (e.g., a tumor cell), and the second scFv is capable of binding to an antigen (e.g., CD3, CD4, CD8, or TCR) on an activated T cell.

[0218] In one aspect, the present invention comprises a BiTE capable of binding to IL13Rα2. In another aspect, the present invention comprises a BiTE capable of binding to CD3 and IL13Rα2. In a particular embodiment, the BiTE comprises any of the antigen-binding domains disclosed herein that are capable of binding to IL13Rα2. In a particular embodiment, the BiTE comprises an antigen-binding domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1 to 22.

[0219] In one aspect, the present invention comprises a BiTE capable of binding to epidermal growth factor receptor (EGFR) or its isoforms (e.g., wild-type EGFR (wtEGFR) or EGFR variant III (EGFRvIII)). In another aspect, the present invention comprises a BiTE capable of binding to CD3 and EGFR or its isoforms. In certain embodiments, the BiTE comprises one of the antigen-binding domains disclosed herein, capable of binding to EGFR or its isoforms. In certain embodiments, the BiTE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54.

[0220] In certain embodiments, BiTE is inducible (e.g., it contains / is driven by an inducible promoter).

[0221] Bispecific constructs including a first CAR and a second CAR (CAR / CAR, see, e.g., Figure 30A), a BiTE and a CAR (BiTE / CAR, see, e.g., Figures 30B-30C), or a first BiTE and a second BiTE (BiTE / BiTE, see, e.g., Figure 30D) are also provided herein.

[0222] A CAR / CAR may include any combination of any of the CARs disclosed herein. In certain embodiments, a CAR / CAR may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:173, and which may be encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:174.

[0223] A BiTE / CAR may include any of the BiTEs disclosed herein, any of the CARs disclosed herein, and any combination thereof. In certain embodiments, a BiTE / CAR includes a BiTE capable of binding to EGFR or its isoform, and a CAR capable of binding to IL13Rα2. In certain embodiments, a BiTE / CAR includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:175, which may be encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:176. In certain embodiments, a BiTE / CAR includes a BiTE capable of binding to IL13Rα2, and a CAR capable of binding to EGFR or its isoform. In certain embodiments, BiTE / CAR may be encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:178, and includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:177.

[0224] A BiTE / BiTE may comprise any of the BiTEs disclosed herein in any combination thereof. In certain embodiments, a BiTE / BiTE comprises a first BiTE capable of binding to EGFR or its isoform, and a second BiTE capable of binding to IL13Rα2. In certain embodiments, a BiTE / BiTE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:179, which may be encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:180.

[0225] E. Nucleic acids and expression vectors This disclosure provides nucleic acids that encode CARs. The nucleic acids of this disclosure may comprise polynucleotide sequences that encode any one of the CARs, BiTEs, BiTE / CARs, or BiTE / BiTEs disclosed herein.

[0226] In one embodiment, the nucleic acid of the present disclosure comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence G VKWAGGSTDYNSALMS (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 7).

[0227] In one embodiment, the nucleic acid encodes a CAR that includes an antigen-binding domain containing 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:57.

[0228] In one embodiment, the nucleic acid encodes a CAR that includes an antigen-binding domain, which includes 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:61.

[0229] In one embodiment, the nucleic acid encodes a CAR that includes an antigen-binding domain comprising 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:57 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:61.

[0230] In one embodiment, the nucleic acid encodes a CAR, which is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence whose antigen-binding domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 133 or 138.

[0231] Also provided is a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), where HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFDV (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), where LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0232] In one embodiment, the nucleic acid encodes a CAR that includes an antigen-binding domain containing 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:67.

[0233] In one embodiment, the nucleic acid encodes a CAR that includes an antigen-binding domain, which includes 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:71.

[0234] In one embodiment, the nucleic acid encodes a CAR comprising an antigen-binding domain including 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:67; and 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:71.

[0235] In one embodiment, the nucleic acid encodes a CAR, which is a single-stranded variable fragment (scFv) encoded by a polynucleotide sequence whose antigen-binding domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 134 or 135.

[0236] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, 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: 57; and 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: 61.

[0237] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, 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:67; and 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:71.

[0238] In another aspect, the present invention provides nucleic acids comprising a 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:75 or SEQ ID NO:76.

[0239] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the first CAR and the second CAR each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain, respectively.

[0240] In a particular embodiment, the antigen-binding domain of the first CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence VKWAGGSTDYNSALMS (SEQ ID NO:2) or GVKWAGGSTDYNSALMS (SEQ ID NO:3), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO:4); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7).

[0241] In a particular embodiment, the antigen-binding domain of the first CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence QGTTALATRFFD (SEQ ID NO: 14); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 16), LCDR2 comprises the amino acid sequence SASYRST (SEQ ID NO: 17), and LCDR3 comprises the amino acid sequence QHHYSAPWT (SEQ ID NO: 18).

[0242] In a particular 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:57; 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:61.

[0243] In a particular 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:67; 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:71.

[0244] In a particular 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:133, SEQ ID NO:134, or SEQ ID NO:135.

[0245] In a particular 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:65 or SEQ ID NO:66 or SEQ ID NO:75 or SEQ ID NO:76.

[0246] In a particular embodiment, the antigen-binding domain of the second CAR comprises a heavy chain variable region comprising three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYSITSDFAWN (SEQ ID NO: 25), HCDR2 comprises the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), and HCDR3 comprises the amino acid sequence VTAGRGFPYW (SEQ ID NO: 27); and a light chain variable region comprising three light chain complementarity-determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO: 28), LCDR2 comprises the amino acid sequence HGINLDD (SEQ ID NO: 143) or HGTNLDD (SEQ ID NO: 29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO: 30).

[0247] In a particular embodiment, the antigen-binding domain of the second CAR includes a heavy 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:31, 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:32. In a particular 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 SEQ ID NO:144, 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:146. In a particular 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 SEQ ID NO:145, 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:147.

[0248] In a particular 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 SEQ ID NO:42, 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:43.

[0249] In certain embodiments, 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:33, SEQ ID NO:141, or SEQ ID NO:41. In certain embodiments, 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:34, SEQ ID NO:142, or SEQ ID NO:44.

[0250] In certain embodiments, the second polynucleotide sequence includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:196. In certain embodiments, 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:36, SEQ ID NO:38, or SEQ ID NO:197.

[0251] A nucleic acid is also provided comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the first CAR is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), where HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID A heavy chain variable region including NO:15); and a light chain variable region including three light chain complementarity-determining regions (LCDRs), wherein LCDR1 includes the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 includes the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 includes the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); the second CAR is a heavy chain variable region including three heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 includes the amino acid sequence GYSITSDFAWN (SEQ ID NO:25), HCDR2 includes the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO:26), and HCDR3 includes the amino acid sequence VTAGRGFPYW (SEQ ID NO: A light chain variable region comprising a heavy chain variable region including NO:27) and three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence HSSQDINSNIG (SEQ ID NO:28), LCDR2 comprises the amino acid sequence HGINLDD (SEQ ID NO:143) or HGTNLDD (SEQ ID NO:29), and LCDR3 comprises the amino acid sequence VQYAQFPWT (SEQ ID NO:30).

[0252] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the first CAR 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: 57 or 67; and 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: 61 or 71; and the second CAR 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: 61 or 71; It 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:139 or 194, and 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:140 or 195.

[0253] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, 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: 138, 133, 134, or 135; 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: 33 or 141.

[0254] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor capable of binding to IL13Rα2, and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, 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: 65 or 66 or 75 or 76; 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: 35 or 196.

[0255] The present invention also comprises a nucleic acid comprising a first polynucleotide sequence encoding a first CAR capable of binding to IL13Rα2, and a second polynucleotide sequence encoding an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-TIM-3 antibody. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0256] Also provided is a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, and a second polynucleotide sequence encoding an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. In certain embodiments, the second polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence encoding SEQ ID NO: 53 or 54. In certain embodiments, the BiTE is capable of binding to wild-type EGFR (wtEGFR). In certain embodiments, the BiTE is capable of binding to EGFR variant III (EGFRvIII).

[0257] In some embodiments, the nucleic acids of the Disclosure are provided for the production of CARs described herein, for example, in mammalian cells. In some embodiments, the nucleic acids of the Disclosure are provided for the amplification of nucleic acids encoding CARs.

[0258] In some embodiments, the nucleic acids of the present disclosure include a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. The linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic sequence or a dicistronic sequence), which are translated as polyproteins that dissociate into separate protein components. For example, a linker for use in the nucleic acids of the present disclosure, including the coding sequence for IL13Rα2 CAR and the coding sequence for EGFR CAR, allows IL13Rα2 CAR and EGFR CAR to be translated as polyproteins that dissociate into separate CARs. In certain embodiments, the nucleic acid includes, in the 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid includes, in the 5' to 3' direction, a second polynucleotide sequence, a linker, and a first polynucleotide sequence.

[0259] In some embodiments, the linker includes 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, of a protein-coding region, thereby resulting in cap-independent translation of the gene. A variety of intrasequence ribosome entry sites are known to those skilled in the art, including, but not limited to, IRES available from viral or cellular mRNA sources, such as 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 IRES available 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 IRES suitable for use in the present invention.

[0260] In some embodiments, the linker includes a nucleic acid sequence encoding a self-cleaving peptide. As used herein, “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that enables encoding multiple proteins as a polyprotein that dissociates into component proteins upon translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving peptides or 2A peptides are known to those skilled in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), zosea signalavirus (TaV), and porcine rhinitis virus-1 (PTV-1); as well as cardioviruses 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 a self-cleaving peptide suitable for use in the present invention.

[0261] 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 processing protein precursors before their secretion. Furin cleaves its consensus recognition sequence at the COOH terminus. Non-limitingly, various furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including Arg-X-Lys-Arg (SEQ ID NO: 117) or Arg-X-Arg-Arg (SEQ ID NO: 118), X2-Arg-X1-X3-Arg (SEQ ID NO: 119), and Arg-X1-X1-Arg (SEQ ID NO: 120) (wherein X1 is any native amino acid, X2 is Lys or Arg, and X3 is Lys or Arg). Those skilled in the art will be able to select a furin cleavage site suitable for use in the present invention.

[0262] 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, a linker containing a nucleic acid sequence encoding furin and F2A, a linker containing a nucleic acid sequence encoding furin and E2A, a linker containing a nucleic acid sequence encoding furin and P2A, and a linker containing a nucleic acid sequence 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. Non-limitingly, various spacer sequences are known in the art, including glycineserine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO: 148) and (GGGS)n (SEQ ID NO: 149) (wherein n represents an integer of at least 1). Exemplary spacer sequences are, non-limitingly, This may include amino acid sequences such as TIFF0007933412000028.tif20158. Those skilled in the art will be able to select a spacer sequence suitable for use in the present invention.

[0263] In some embodiments, the nucleic acids of this disclosure may 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.

[0264] In some embodiments, the nucleic acid encoding the external CAR is functionally ligated to the promoter. In some embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0265] Promoter suitable for expression in bacterial cells includes, but is not limited to, lacI, lacZ, T3, T7, gpt, lambdaP, and trc. Promoter suitable for expression in eukaryotic cells includes, but is 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-chain terminal repeat sequences derived from retroviruses; 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 may be isolated from and derived from numerous 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 based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulating promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) response 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 induction promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.)), photoregulating promoters, synthesis-inducing promoters, etc.).

[0266] 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. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI(p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117:1565.

[0267] Suitable promoters for expression in yeast cells are 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 Pichia). The selection of appropriate vectors and promoters is well within the realm of those skilled in the art. Promoter suitable 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, etc., in vivo regulatory promoters or related promoters (see, for example, 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), etc. (see, 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 (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); quiescent promoter, e.g., dps promoter, spv promoter, etc.; promoters derived from pathogenic island SPI-2 (see, e.g., WO96 / 17951); actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in See Molecular and 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 powerful promoters 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 its conformation when in 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 (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0268] Other examples of suitable promoters include the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of promoting high levels of expression of any polynucleotide sequence functionally ligated to it. Other constitutive promoter sequences may also be used, including, but not limited to, the monkey virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV) or human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, EF-1 alpha promoter, and human gene promoters such as actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. 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 an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence functionally ligated to it when such expression is desired, or turn off the expression when such expression is undesirable. Examples of inductive promoters include, but are not limited to, metallotionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In certain embodiments, the present invention provides polynucleotide sequences encoding CARs (e.g., bispecific CARs, BiTEs, tandem CARs, parallel CARs, and others) comprising an inductive promoter. In certain embodiments, 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. In certain embodiments, the inductive promoter comprises a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:161.In certain embodiments, the inducible promoter contains a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:198. In certain embodiments, the sequence containing SEQ ID NO:198 is repeated to enhance the expression level of T cells. For example, in certain embodiments, the inducible promoter may contain a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:162.

[0269] In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched via induction of an induction system. Systems suitable 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 generate irreversibly switchable promoters. Methods, mechanisms, and requirements for performing site-directed recombination, as described elsewhere in this specification, are used to generate irreversibly switched promoters and are well known in the art. For example, see Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), which are incorporated herein by reference.

[0270] In some embodiments, the nucleic acids of the Disclosure further comprise nucleic acid sequences encoding a CAR-induced expression cassette. In one embodiment, the CAR-induced 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 nucleic acid sequences encoding a cytokine functionally ligated to a T cell activation response promoter. In some embodiments, the cytokine functionally ligated to the T cell activation response promoter resides on separate nucleic acid sequences. In one embodiment, the cytokine is IL-12.

[0271] 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 replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those skilled in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided as examples, but should not be construed as limiting in any way: 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). Prokaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0272] Expression vectors generally provide insertion of nucleic acid sequences encoding heterologous proteins, often with convenient restriction sites located near the promoter sequence. Selective markers that operate in the expression host may exist. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (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: See 1088-1097; International Publication Nos. 94 / 12649, 93 / 03769; 93 / 19191; 94 / 28938; 95 / 11984 and 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; Srivastava, Samulski et al., J. Vir. (1989) 63: 3822-3828 in International Publication No. 93 / 09239; 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; viral vectors based on 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), etc., but not limited to these.

[0273] Additional expression vectors suitable for use include, but are not limited to, lentiviral vectors, gamma-retroviral vectors, foamy viral vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid viral vectors, and transposon-mediated vectors. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0274] Generally, a suitable vector contains a functional origin of replication, a promoter sequence, a favorable restriction endonuclease site, and one or more selection markers in at least one organism (e.g., International Publication No. 01 / 96584; 01 / 29058; and U.S. Patent No. 6,326,193).

[0275] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce CAR into immune cells or their progenitor cells (e.g., T cells). Therefore, the expression vector (e.g., a lentiviral vector) of the present invention may contain a nucleic acid encoding the CAR. In some embodiments, the expression vector (e.g., a lentiviral vector) will contain additional elements that facilitate the functional expression of the CAR encoded therein. In some embodiments, the expression vector containing the nucleic acid encoding the 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 downstream transgene expression (e.g., the nucleic acid sequence encoding the CAR). Physiological promoters (e.g., EF-1α promoters) may be less likely to induce integration-mediated genotoxicity and may counteract 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 includes non-essential cis-acting sequences that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polyprint lactate and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear transport. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vector of the present invention (e.g., a lentiviral vector). In some embodiments, the vector further includes post-transcriptional regulatory elements. Post-transcriptional regulatory elements may 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 includes a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vector of the present invention (e.g., a lentiviral vector).The vectors of the present invention may further include additional 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 necessary for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and 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.

[0276] 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 can infect and then be incorporated into the host genome (e.g., mammalian genome) only once, but cannot be further passaged. Therefore, self-inactivating vectors can significantly reduce the risk of producing replication-eligible viruses.

[0277] In some embodiments, the nucleic acid of the present invention may be RNA, for example, in vitro synthesized RNA. Methods for the in vitro synthesis of RNA are known to those skilled in the art; RNA containing the CAR encoding sequence of the present disclosure can be synthesized using any known method. 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 or ex 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.

[0278] Expression vectors to be introduced into cells to evaluate the expression of polypeptides or portions thereof may also contain one or both selection marker genes and / or reporter genes to facilitate the identification and selection of cells expressing the polypeptide from a cell population intended for transfection or infection with the viral vector. In some embodiments, the selection marker may be vested on a separate DNA fragment and used in co-transfection procedures. Both the selection marker and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0279] 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 is neither present in nor expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is revealed by several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is evaluated at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0280] F. Modified immune cells The present invention provides modified immune cells or their progenitor cells (e.g., T cells) comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2 (e.g., human IL13Rα2 or canine IL13Rα2). Modified immune cells or their progenitor cells comprising BiTE, BiTE / BiTE, or BiTE / CAR are also provided. The present invention also comprises modified immune cells or their progenitor cells comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0281] One aspect of the present invention provides a modified immune cell or its progenitor cell containing a CAR capable of binding to IL13Rα2, wherein the CAR comprises a heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs). HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO: 1) or SRNGMS (SEQ ID NO: 12), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15). The CAR also comprises a light chain variable region containing three light chain complementarity-determining regions (LCDRs). LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), 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).

[0282] Another aspect of the present invention comprises modified immune cells or their progenitor cells containing a CAR capable of binding to IL13Rα2, wherein the CAR comprises 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 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 or 20.

[0283] Modified immune cells or their progenitor cells containing a CAR capable of binding to IL13Rα2 are also provided, wherein the 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.

[0284] In another aspect, the present invention provides modified immune cells or their progenitor cells containing a CAR capable of binding to IL13Rα2, 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:21 or 22.

[0285] Another aspect of the present invention includes modified immune cells or their progenitor cells comprising a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain capable of binding to IL13Rα2; and a second chimeric antigen receptor (CAR) comprising a second antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR) or its isoform.

[0286] A further aspect of the present invention includes a modified immune cell or its progenitor cell comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to the epidermal growth factor receptor (EGFR) or its isoform, wherein 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), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (SEQ ID NO: 3) or TVSSGGSYIYYADSVKG (SEQ ID NO: 13), and HCDR3 comprises the amino acid sequence DHRDAMDY (SEQ ID NO: 4) or QGTTALATRFFDV (SEQ ID NO: 15). The first CAR also comprises a light chain variable region comprising three light chain complementarity-determining regions (LCDRs). LCDR1 contains the amino acid sequence TASLSVSSTYLH (SEQ ID NO: 5) or KASQDVGTAVA (SEQ ID NO: 16), 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), HCDR2 contains the amino acid sequence GYISYSGNTRYNPSLK (SEQ ID NO: 26), 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).

[0287] Another aspect of the present invention includes modified immune cells or progenitor cells comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein the first CAR comprises a heavy 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: 8 or 19, and 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 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:42 or 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:32 or SEQ ID NO:43 or SEQ ID NO:146 or SEQ ID NO:147.

[0288] Modified immune cells or their progenitor cells are also provided, comprising a first chimeric antigen receptor capable of binding to IL13Rα2, and a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR) or its isoform, wherein 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 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 an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:34 or SEQ ID NO:44.

[0289] In certain embodiments, 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 D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F It can be bound to an EGFR isoform selected from the group consisting of EGFR variant II, or any combination thereof.

[0290] Modified cells may further contain immune checkpoint inhibitors, in which case the modified cells secrete immune checkpoint inhibitors. Immune checkpoints include, but are not limited to, CTLA-4, PD-1, and TIM-3. Immune checkpoint inhibitors include, but are not limited to, anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-TIM-3 antibodies.

[0291] The modified cells may further contain an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoform. The modified cells secrete the BiTE. In certain embodiments, the inducible BiTE contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 54. In certain embodiments, the BiTE is capable of binding to wild-type EGFR (wtEGFR). In certain embodiments, the BiTE is capable of binding to EGFR variant III (EGFRvIII).

[0292] G. Sources of immune cells In some embodiments, the source of immune cells (e.g., T cells) for ex vivo manipulation is obtained from the subject. The source of target 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 by the modified immune cells of the present invention, and may be, for example, 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.

[0293] Immune cells can be obtained from several 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, such as lymphocytes, typically myeloid or lymphoid cells including T cells and / or NK cells. Other exemplary cells include stem cells such as induced pluripotent stem cells (iPSCs), as well as multipotent and pluripotent stem cells. In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or self. The cells are typically primary cells, e.g., primary cells isolated directly from the subject and / or isolated and frozen from the subject.

[0294] In certain 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, lymphoid 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, cells generated from a subject and manipulated to alter (e.g., induce mutations in) the expression of one or more target genes, such as 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, lymphoid progenitor cells, or hematopoietic stem cells differentiated from iPS cells.

[0295] In some embodiments, cells include one or more subsets of T cells or other cell types, e.g., entire T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, defined, e.g., function, activation state, maturation, differentiation potential, growth, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in specific organs or compartments, 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, among others, naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, e.g., stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), 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, innate 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 certain embodiments, several T cell lines available in the art may be used.

[0296] In some embodiments, the method includes the steps of isolating immune cells from a subject, and preparing, processing, culturing, 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, such as 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 a subject requiring or to be administered cell therapy. In some embodiments, the subject is a human being in need of a specific therapeutic intervention, such as 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, 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, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, including processed samples derived therefrom.

[0297] In some contexts, the sample from which cells are derived or isolated is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. 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 tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. Samples include autologous and allogeneic sources in connection with cell therapy, such as adoptive cell therapy.

[0298] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from heterogeneous sources, such as mice, rats, non-human primates, and pigs. In some embodiments, cell isolation includes one or more preparation steps and / or cell separation steps that are not based on affinity. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to remove, for example, unwanted 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 to and / or resistance to a particular component.

[0299] In some cases, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. The sample contains, in some aspects, lymphocytes including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects, 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 place the cells in a buffer or medium suitable for the subsequent processing step. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing step is accomplished by tangent flow filtration (TFF) as described by the manufacturer. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in a culture medium. In some embodiments, the method includes density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and performing centrifugation by Percoll or Ficoll gradient.

[0300] In one embodiment, the immune cells are cells obtained from the circulating blood of an individual obtained by apheresis or leukocyte apheresis. Apheresis products typically contain 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 to place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the washing solution may be calcium-deficient and magnesium-deficient, or deficient in many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free and Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in a culture medium.

[0301] 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, such as surface markers, such as surface proteins, intracellular markers, or nucleic acids, within the cells. In some embodiments, any known method may be used for separation based on such markers. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the separation includes the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, by incubation with an antibody or binding partner that specifically binds to such markers, generally followed by a washing step, and the separation of cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0302] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or negative selection, where cells not bound to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some situations, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are unavailable, so that 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 cells expressing a particular cell population or a particular marker. For example, positive selection or enrichment of a particular type of cell, e.g., cells expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in the complete absence of cells not expressing 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 percentage of such cells, but does not need to result in the complete removal of all such cells.

[0303] In some cases, multiple separation steps are performed, in which fractions positively or negatively selected from one step are subjected to further separation steps, such as subsequent positive or negative selection. In some cases, cells expressing multiple markers can be depleted in a single separation step by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, positive selection of multiple cell types can be performed simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0304] In some embodiments, one or more T cell populations are positive for one or more specific markers, such as surface markers (marker+), or express them at high levels (marker). high ) cells, or negative for one or more markers (marker -) or express it at a relatively low level (marker low ) Cells are enriched or depleted. For example, in some aspects, cells that are positive for or express at 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 (CD8+ cells, or T cells, e.g., CD3+ cells) are enriched (i.e., positively selected) for being positive for or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or depleted (i.e., negatively selected) for being positive for or expressing high surface levels of CD45RA. In some embodiments, cells are enriched or depleted for being positive for or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, CD8+ T cells are enriched for being positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0305] 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, a CD4+ or CD8+ selection step is used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive, memory, 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, for example, on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase efficacy, for example, to improve long-term survival, growth, and / or engraftment after administration, and it is particularly robust in some aspects in such subpopulations. In some embodiments, the efficacy is further enhanced by combining TCM-enriched CD8+ T cells and CD4+ T cells.

[0306] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions using anti-CD8 antibodies and anti-CD62L antibodies, etc. In some embodiments, the enriched subpopulation is the CD4+ T cell population and the CD8+ T cell subpopulation, e.g., central memory (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positivity or high surface expression for CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some embodiments, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, the isolation of a CD8+ population enriched with TCM cells is carried out by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, as well as positive selection based on CD62L. Such selections are carried out simultaneously in some aspects and sequentially in other aspects. In some aspects, the same CD4 expression-based selection step used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, thereby retaining and using both the positive and negative fractions from the CD4-based isolation in subsequent steps of the method, optionally following one or more further positive or negative selection steps.

[0307] CD4+ T helper cells are sorted into naive, central memory, 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, CD11b, 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.

[0308] In some embodiments, cells are incubated and / or cultured before or in connection with genetic engineering. The incubation step may include culture, cultivation, stimulation, activation, and / or proliferation. In some embodiments, a composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce proliferation, growth, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic engineering, e.g., introduction of recombinant antigen receptors. Conditions may include one or more of a specific medium, 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, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the activator initiates or starts the TCR / CD3 intracellular signaling cascade in T cells. Such activators may include, for example, antibodies conjugated to a solid support such as beads, for example antibodies specific to TCR components and / or costimulatory receptors, such as anti-CD3, anti-CD28, and / or one or more cytokines. Optionally, the amplification method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (for example, at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0309] In another e...

Claims

1. A polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR) that specifically binds to IL13Rα2, wherein the CAR comprises an IL13Rα2 antigen-binding domain, a hinge domain, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain, wherein the IL13Rα2 antigen-binding domain is scFv, the hinge domain and the transmembrane domain are derived from human CD8 alpha, the intracellular costimulatory signaling domain is derived from human 4-1BB, the intracellular signaling domain is derived from human CD3 zeta, and the nucleic acid comprises a sequence of SEQ ID NO: 133 encoding scFv.

2. The polynucleotide according to claim 1, wherein the nucleic acid encoding the hinge domain is the sequence of SEQ ID NO:

45.

3. The polynucleotide according to claim 1 or 2, wherein the nucleic acid encoding the transmembrane domain is the sequence of SEQ ID NO:

46.

4. The polynucleotide according to any one of claims 1 to 3, wherein the nucleic acid encoding the intracellular costimulatory signaling domain is the sequence of SEQ ID NO:

47.

5. The polynucleotide according to any one of claims 1 to 4, wherein the nucleic acid encoding the intracellular signaling domain is the sequence of SEQ ID NO:

48.

6. The polynucleotide according to any one of claims 1 to 5, further comprising a nucleic acid sequence encoding a human CD8 alpha-signal peptide, wherein the nucleic acid encoding the CAR further comprises a nucleic acid sequence encoding a human CD8 alpha-signal peptide.

7. The polynucleotide according to claim 6, wherein the nucleic acid sequence encoding the human CD8 alpha signal peptide is the sequence of SEQ ID NO:

49.

8. The polynucleotide according to claim 1, wherein the nucleic acid encoding the CAR comprises a nucleic acid sequence of SEQ ID NO:45 encoding the hinge domain, a nucleic acid sequence of SEQ ID NO:46 encoding the transmembrane domain, a nucleic acid sequence of SEQ ID NO:47 encoding the intracellular costimulatory signaling domain, and a nucleic acid sequence of SEQ ID NO:48 encoding the intracellular signaling domain.

9. The polynucleotide according to claim 8, wherein the nucleic acid encoding the CAR comprises nucleotides 70 to 1461 of SEQ ID NO:

66.

10. The polynucleotide according to claim 9, wherein the nucleic acid encoding the CAR includes a nucleic acid sequence of SEQ ID NO: 49 encoding a human CD8 alpha signal peptide upstream of nucleotides 70 to 1461 of SEQ ID NO:

66.

11. (a) A first nucleic acid encoding a first chimeric antigen receptor (CAR) that specifically binds to IL13Rα2, wherein the CAR comprises an IL13Rα2 antigen-binding domain, a hinge domain, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain, wherein the IL13Rα2 antigen-binding domain is scFv, and the first nucleic acid comprises a sequence of SEQ ID NO: 133 encoding scFv, (b) A second nucleic acid encoding a second CAR that specifically binds to the epidermal growth factor receptor (EGFR) or its isoform, wherein the CAR comprises an EGFR antigen-binding domain, a hinge domain, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain, the EGFR antigen-binding domain being scFv, and the second nucleic acid comprising a sequence of SEQ ID NO: 141 encoding scFv, A polynucleotide containing, A polynucleotide in which the hinge domain and transmembrane domain of the first CAR and the second CAR are both derived from human CD8 alpha, the intracellular co-stimulatory signaling domains of the first CAR and the second CAR are both derived from human 4-1BB, and the intracellular signaling domains of the first CAR and the second CAR are both derived from human CD3 zeta.

12. The polynucleotide according to claim 11, wherein the nucleic acid encoding each hinge domain is the sequence of SEQ ID NO:

45.

13. The polynucleotide according to claim 11 or 12, wherein the nucleic acid encoding each transmembrane domain is the sequence of SEQ ID NO:

46.

14. The polynucleotide according to any one of claims 11 to 13, wherein the nucleic acid encoding each intracellular costimulatory signaling domain is the sequence of SEQ ID NO:

47.

15. The polynucleotide according to any one of claims 11 to 14, wherein the nucleic acid encoding each intracellular signaling domain is the sequence of SEQ ID NO:

48.

16. The polynucleotide according to any one of claims 11 to 15, wherein both nucleic acids encoding the first CAR and the second CAR further comprise nucleic acid sequences encoding human CD8 alpha-signal peptide.

17. The polynucleotide according to claim 16, wherein the nucleic acid sequence encoding the human CD8 alpha signal peptide of the first CAR is the sequence of SEQ ID NO:

49.

18. The polynucleotide according to claim 11, wherein the first nucleic acid encoding the first CAR and the second nucleic acid encoding the second CAR each comprise a nucleic acid sequence of SEQ ID NO:45 encoding the hinge domain, a nucleic acid sequence of SEQ ID NO:46 encoding the transmembrane domain, a nucleic acid sequence of SEQ ID NO:47 encoding the intracellular costimulatory signaling domain, and a nucleic acid sequence of SEQ ID NO:48 encoding the intracellular signaling domain.

19. The polynucleotide according to claim 18, wherein the first nucleic acid encoding the first CAR comprises nucleotides 70 to 1461 of SEQ ID NO:

66.

20. The polynucleotide according to claim 19, wherein the first nucleic acid encoding the first CAR includes a nucleic acid sequence of SEQ ID NO: 49 encoding a human CD8 alpha signal peptide upstream of nucleotides 70-1461 of SEQ ID NO:

66.

21. The polynucleotide according to any one of claims 16 to 20, wherein the second nucleic acid encoding the second CAR further comprises a nucleic acid sequence encoding a human CD8 alpha-signal peptide having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:

49.

22. The polynucleotide according to any one of claims 11 to 21, wherein the first nucleic acid encoding the first CAR and the second nucleic acid encoding the second CAR are separated by a nucleic acid encoding a linker containing a 2A peptide.

23. The polynucleotide according to claim 22, wherein the 2A peptide is a P2A peptide.

24. The polynucleotide according to claim 22 or 23, wherein the polynucleotide comprises, in the 5' to 3' direction, a first nucleic acid encoding the first CAR, a nucleic acid encoding the linker, and a second nucleic acid encoding the second CAR.

25. A vector comprising a polynucleotide according to any one of claims 1 to 24.

26. The vector according to claim 25, which is a lentiviral vector.

27. ​​The vector according to claim 25 or 26, comprising an elongation factor-1-alpha promoter.

28. A human immune cell, which is a T cell, NK cell, or NKT cell, comprising a polynucleotide according to any one of claims 1 to 24, or a vector according to any one of claims 25 to 27.

29. The human immune cell according to claim 28, which is a human T cell.

30. A pharmaceutical composition comprising a population of human immune cells according to claim 28 or 29 and a pharmaceutically acceptable carrier.

31. A composition for use in treating cancer in a human subject in need thereof, wherein the composition comprises a therapeutically effective amount of human immune cells according to claim 28 or 29 or the pharmaceutical composition according to claim 30.

32. The composition according to claim 31, wherein the cancer is a glioma, an astrocytoma, a high-grade astrocytoma, or a glioblastoma.

33. The composition according to claim 31 or 32, wherein the composition is administered in combination with an additional therapeutic agent.

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