Synthetic CARs for treating IL13Rα2-positive human and canine tumors

Abstract

The present disclosure provides engineered immune cells or their precursors (e.g., T cells) comprising a chimeric antigen receptor (CAR) capable of binding to human IL13Rα2. Bispecific CARs, parallel CARs, tandem CARs, BiTEs, BiTE / CARs, and BiTE / BiTEs are also provided. Compositions and methods of treatment are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 892,114, filed August 27, 2019, 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 glioblastomas (GBM), are the most common primary malignant brain tumors and are associated with high morbidity and mortality. The aggressive nature of invasive growth of glioma cells within the central nervous system (CNS) makes total resection unattainable. Despite the best available treatments, including surgical resection, radiation therapy, chemotherapy, and tumor-treating electric fields, median survival is only 12–17 months for patients with GBM and 2–5 years for patients with grade III gliomas.

[0003] Adoptive immunotherapy with redirected T cells is a viable strategy for treating these malignancies. Long-term disease-free survival was achieved in patients with refractory chronic lymphocytic leukemia after treatment with CD19-targeted chimeric antigen receptor-modified autologous T (CAR T) cells, and complete remission was achieved in 90% of patients with relapsed acute lymphoblastic leukemia (ALL) with this strategy. 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 utilized in a phase I clinical trial (NCT02209376) in 10 patients with recurrent GBM. After CAR T cell infusion, there were clear changes in the tumor microenvironment, including a reduction in EGFRvIII target antigens associated with CAR T cell trafficking and in situ functional activation. However, this trial was not powered to determine 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 zetakine, a mutant IL13 cytokine fused with a T cell signaling domain, in one patient with recurrent multifocal GBM, which led to complete tumor regression over a 7.5 month period.

[0004] Interleukin-13 receptor α2 (IL13Rα2) is expressed in various human tumor types but is not expressed in normal human tissues, except in adult testis (Figure 7B). IL13 signaling via IL13Rα2 plays an important role in cell migration and invasion. Previous studies have found that 82% of GBM cases expressed IL13Rα2. Neutralizing and drug-conjugated antibodies targeting IL13Rα2 inhibited tumor growth in xenograft mouse models. IL13Rα2-based tumor vaccines have also been beneficial in pediatric glioma patients. Although IL13 zetakine-redirected T cells bound to IL13Rα2 and induced limited clinical responses, these T cells also bound to IL13Rα1, which is expressed in several normal human tissues (Figure 7A), demonstrating deleterious off-target effects.

[0005] The tumor microenvironment of malignant glioma is immunosuppressive, as demonstrated after infusion of CAR T cells. Immune checkpoint receptors (e.g., PD-1, CTLA-4, TIM-3, and LAG-3) are a set of molecules that downregulate activated T cell stimulation with distinct spatiotemporal profiles to regulate T cell function. Checkpoint inhibitors have been applied in cancer therapy to overcome T cell inhibition within the immunosuppressive tumor microenvironment and recruit 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 involving engineered T cells.

[0006] There is a need in the art for compositions and methods for treating IL13Rα2-positive tumors. The present invention addresses and satisfies this need. Summary of the Invention

[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): 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): 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 comprises a heavy chain variable region comprising an amino acid sequence 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 comprises a light chain variable region comprising an amino acid sequence 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 comprises a heavy chain variable region comprising 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 comprising an amino acid sequence 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 antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0010] In certain embodiments, the antigen-binding domain is a single-chain 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.

[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): 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): 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 comprises a heavy chain variable region comprising an amino acid sequence 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 comprises a light chain variable region comprising an amino acid sequence 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 comprises a heavy chain variable region comprising an amino acid sequence 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 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 antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0014] In certain embodiments, the antigen-binding domain is a single-chain 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:21 or 22.

[0015] In certain embodiments, the CAR is capable of binding to IL13Rα2. In certain embodiments, the CAR is capable of binding to human IL13Rα2. In certain embodiments, the CAR is capable of binding to canine IL13Rα2. In certain embodiments, the CAR is capable of binding to human IL13Rα2 and canine IL13Rα2.

[0016] In certain embodiments, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T cell receptor, 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). In certain embodiments, the transmembrane domain comprises 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 comprises a costimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in 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 comprises the costimulatory domain of 4-1BB. In certain embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof. In certain embodiments, the intracellular signaling domain comprises 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 comprising 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 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 a nucleic acid comprising a polynucleotide sequence encoding any of the CARs contemplated 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, the CAR 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): 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): 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 comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57. In certain embodiments, the antigen-binding domain comprises a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61. In certain embodiments, the antigen-binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:57; and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:61.

[0024] In certain embodiments, the antigen-binding domain is a single-chain 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, the CAR 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): 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): 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 comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67. In certain embodiments, the antigen-binding domain comprises a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71. In certain embodiments, the antigen-binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67; and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71.

[0027] In certain embodiments, the antigen-binding domain is a single-chain 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 comprises the transmembrane domain of CD8 alpha. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the costimulatory signaling domain comprises the costimulatory domain of 4-1BB. In certain embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta.

[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, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:57; and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: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 a nucleic acid 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 an isoform thereof, wherein the first CAR and the second CAR each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0033] In certain embodiments, 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 certain embodiments, the antigen binding domain of the first CAR comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:57; and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:61.

[0035] In certain embodiments, the antigen-binding domain of the first CAR is a single-chain 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 certain embodiments, 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 certain embodiments, the antigen binding domain of the first CAR comprises a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67; and a light chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71.

[0038] In certain embodiments, the antigen-binding domain of the first CAR is a single-chain 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 certain embodiments, 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.

[0040] In certain embodiments, 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 comprises a heavy chain variable region comprising an amino acid sequence 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 comprises a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In certain embodiments, the antigen-binding domain of the second CAR comprises a heavy chain variable region comprising an amino acid sequence 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 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-chain 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 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 SEQ ID NO:35 or SEQ ID NO:196.

[0044] In certain embodiments, the transmembrane domain of the first CAR and / or the second CAR is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T cell receptor, 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). In certain embodiments, the transmembrane domain of the first CAR and / or the second CAR comprises the transmembrane domain of CD8 alpha.

[0045] In certain embodiments, the intracellular domain of the first CAR and / or the second CAR comprises a costimulatory signaling domain and an intracellular signaling domain. In certain embodiments, 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 in 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 of the first CAR and / or the second CAR comprises the costimulatory domain of 4-1BB.

[0046] In certain embodiments, the intracellular signaling domain of the first CAR and / or the second CAR comprises an intracellular domain selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof. In certain embodiments, the intracellular signaling domain of the first CAR and / or the second CAR comprises 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 an isoform thereof. The first CAR 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) 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:17). and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR comprises 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 an isoform thereof. 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 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: 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 an isoform thereof. The first CAR comprises a single-chain 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-chain 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 an isoform thereof, wherein the first polynucleotide sequence comprises a sequence 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 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 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.

[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 an isoform thereof.

[0054] 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.

[0055] In certain embodiments, a BiTE is capable of binding to wild-type EGFR (wtEGFR). In certain embodiments, a BiTE is capable of binding to EGFR variant III (EGFRvIII).

[0056] In certain embodiments, the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker. In certain embodiments, the linker comprises an internal ribosome entry site (IRES) or a nucleotide sequence encoding a self-cleaving peptide. In certain embodiments, the self-cleaving peptide is a 2A peptide. In certain embodiments, the 2A peptide is selected from the group consisting of porcine teschovirus-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 comprises a furin cleavage site.

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

[0058] In certain embodiments, the nucleic acid further comprises an inducible promoter, wherein the inducible promoter comprises a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 161, 162, or 198.

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

[0060] In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector. 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 an engineered immune cell or a precursor thereof comprising any of the CARs contemplated herein, any of the nucleic acids contemplated herein, or any of the vectors contemplated herein.

[0062] In another aspect, the present invention provides modified immune cells or precursors thereof comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2. A 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:17). and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18).

[0063] In another aspect, the present invention provides an engineered immune cell or a precursor thereof comprising a CAR capable of binding to IL13Rα2, wherein the CAR comprises a heavy chain variable region comprising 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 comprising an amino acid sequence 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 an engineered immune cell or a precursor thereof comprising a CAR capable of binding to IL13Rα2, wherein the CAR comprises a single chain 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.

[0065] In another aspect, the present invention provides an engineered immune cell or progenitor thereof comprising a 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.

[0066] In certain embodiments, the CAR is capable of binding to IL13Rα2. In certain embodiments, the CAR is capable of binding to human IL13Rα2.

[0067] In certain embodiments, the modified cells further comprise an immune checkpoint inhibitor, wherein 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 engineered cells further comprise an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, wherein the engineered cells secrete the BiTE. In certain embodiments, the inducible BiTE comprises an amino acid sequence 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 an engineered immune cell, or a precursor thereof, 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 an isoform thereof.

[0070] In another aspect, the present invention provides a modified immune cell, or a precursor 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. The first CAR 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) 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:17). and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR comprises 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 an engineered immune cell or progenitor thereof comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof. The first CAR comprises a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20. The second CAR comprises a heavy chain variable region comprising an amino acid sequence 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 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 an engineered immune cell or a precursor 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 a single-chain variable fragment (scFv) comprising an amino acid sequence 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-chain variable fragment (scFv) comprising an amino acid sequence 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 an engineered immune cell or a progenitor 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or 24; and the second CAR comprises an amino acid sequence 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 cells further comprise an immune checkpoint inhibitor, wherein 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.

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

[0076] In certain embodiments of the modified cells, the second CAR is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, and EGFR. A289V , EGFR A289D , EGFR A289T , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F and EGFR variant II, or any combination thereof.

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

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

[0079] In another aspect, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the modified cells contemplated herein or any of the pharmaceutical compositions contemplated herein.

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

[0081] In another aspect, the present invention provides a method of treating glioblastoma in a subject in need thereof, the method comprising administering to the subject an effective amount of modified T cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2. A 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:17). and LCDR3 comprises 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 of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the CAR comprises a heavy chain variable region comprising 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 comprising 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 of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the CAR comprises a single chain 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 SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:22.

[0084] In another aspect, the present invention provides a method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T 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:23 or SEQ ID NO:24 or SEQ ID NO:55 or SEQ ID NO:56.

[0085] In certain embodiments, the method further comprises administering an immune checkpoint inhibitor, wherein 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. In certain embodiments, the immune checkpoint inhibitor is co-administered with the modified T cells.

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

[0087] In certain embodiments, the inducible BiTE comprises an amino acid sequence 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). In certain embodiments, the BiTE is co-administered with engineered T cells.

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

[0089] In another aspect, the present invention provides a method for treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T 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 an isoform thereof.

[0090] In another aspect, the present invention provides a method of treating glioblastoma in a subject in need thereof, comprising administering to the 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 an isoform thereof. The first CAR 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) 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:17). and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18). The second CAR comprises 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 of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of engineered 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 an isoform thereof. The first CAR comprises a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 20. The second CAR comprises a heavy chain variable region comprising an amino acid sequence 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 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 of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of engineered 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 an isoform thereof. The first CAR comprises a single-chain variable fragment (scFv) comprising an amino acid sequence 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-chain variable fragment (scFv) comprising an amino acid sequence 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 of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of engineered 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 an isoform thereof. The first CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23 or 24; and the second CAR comprises an amino acid sequence 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 comprises administering an immune checkpoint inhibitor, wherein 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. In certain embodiments, the immune checkpoint inhibitor is co-administered with the modified cells.

[0095] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a CAR comprising 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. 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 an isoform thereof. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 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, and the first CAR and the second CAR are separated by a cleavable linker. In certain embodiments, the parallel CAR comprises an amino acid sequence 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 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 a BiTE and a CAR. In certain embodiments, the BiTE comprises an antigen-binding domain capable of binding to EGFR or an isoform thereof, and the CAR comprises an antigen-binding domain capable of binding to IL13Rα2. In certain embodiments, the BiTE comprises an antigen-binding domain capable of binding to IL13Rα2, and the CAR comprises an antigen-binding domain capable of binding to EGFR or an isoform thereof. 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 nucleic acids comprising polynucleotide sequences encoding a first BiTE and a second BiTE. In certain embodiments, 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 an isoform thereof. 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 179. [The present 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 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 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); Including, CAR. [The present invention 1002] 1001. A CAR of the present invention, 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. [The present invention 1003] The CAR of the present invention 1001 or 1002, wherein the antigen-binding domain comprises 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. [The present invention 1004] Any of the aforementioned CARs of the present invention, 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. [The present invention 1005] Any of the aforementioned CARs of the present invention, wherein the antigen-binding domain is selected from the group consisting of a full-length antibody or antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. [The present invention 1006] Any of the aforementioned CARs of the present invention, wherein the antigen-binding domain is a single-chain 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 present 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 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 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); Including, CAR. [The present invention 1008] 1007. The CAR of the present invention, 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:19. [The present invention 1009] The CAR of the present invention 1007 or 1008, wherein the antigen-binding domain comprises 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. [The present invention 1010] A CAR of any of claims 1007 to 1009, 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: 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. [The present invention 1011] The CAR of any of claims 1007 to 1010, wherein the antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. [The present invention 1012] Any of the CARs of claims 1007 to 1011, wherein the antigen-binding domain is a single-chain 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: 21 or 22. [The present invention 1013] The CAR of any of claims 1001 to 1012, which is capable of binding to IL13Rα2. [The present invention 1014] The CAR of any of the present inventions 1001 to 1013, which is capable of binding to human IL13Rα2. [The present invention 1015] The CAR of any of the present inventions 1001 to 1014, which is capable of binding to canine IL13Rα2. [The present invention 1016] The CAR of any of the present inventions 1001 to 1015, which is capable of binding to human IL13Rα2 and canine IL13Rα2. [The present invention 1017] 10. The CAR of any of claims 1001 to 1016, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, 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). [The present invention 1018] A CAR of any of 1001 to 1017, wherein the transmembrane domain comprises the transmembrane domain of CD8. [The present invention 1019] A CAR of the present invention, wherein the transmembrane domain of CD8 is the transmembrane domain of CD8 alpha. [The present invention 1020] The CAR of any of 1001 to 1019, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. [The present invention 1021] The CAR of any of claims 1001 to 1020, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in 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). [The present invention 1022] A CAR of the present invention 1021, wherein the intracellular domain comprises the costimulatory domain of 4-1BB. [The present invention 1023] CAR of any of 1020 to 1022, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof. [The present invention 1024] A CAR of any of 1020 to 1023, wherein the intracellular signaling domain comprises the intracellular domain of CD3ζ. [The present 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 is a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 Including, CAR. [The present 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 is a heavy chain variable region comprising an amino acid sequence 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:20 Including, CAR. [The present 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 or SEQ ID NO:24 or SEQ ID NO:55 or SEQ ID NO:56. [The present invention 1028] A nucleic acid comprising a polynucleotide sequence encoding any one of CARs 1001 to 1028 of the present invention. [The present invention 1029] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain 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 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); A nucleic acid comprising: [The present invention 1030] 1029. The nucleic acid of the invention, 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. [The present invention 1031] 10. The nucleic acid of claim 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. [The present invention 1032] The nucleic acid of any of claims 1029 to 1031, 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. [The present invention 1033] The nucleic acid of any of claims 1029 to 1032, wherein the antigen-binding domain is a single-chain 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. [The present invention 1034] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain 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 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); A nucleic acid comprising: [This invention 1035] 1034. The nucleic acid of the invention, 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. [The present invention 1036] The nucleic acid of 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. [This invention 1037] The nucleic acid of any of claims 1034 to 1036, 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. [The present invention 1038] The nucleic acid of any of claims 1034 to 1037, wherein the antigen-binding domain is a single-chain 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. [This invention 1039] 1039. The nucleic acid of any one of claims 1029 to 1038, wherein the transmembrane domain comprises the transmembrane domain of CD8 alpha. [The present invention 1040] The nucleic acid of any of claims 1029 to 1039, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. [The present invention 1041] 1040. The nucleic acid of claim 10, wherein the costimulatory signaling domain comprises the costimulatory domain of 4-1BB. [The present invention 1042] The nucleic acid of claim 1040 or 1041, wherein the intracellular signaling domain comprises the intracellular domain of CD3ζ. [This invention 1043] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain is a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: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 A nucleic acid comprising: [This invention 1044] A nucleic acid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, The antigen-binding domain is a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: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 A nucleic acid comprising: [This invention 1045] A nucleic acid 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. [The present 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; wherein the first CAR and the second CAR each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain. [This 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 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); 1046. The nucleic acid of the present invention, comprising: [This invention 1048] The nucleic acid of 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. [This invention 1049] The nucleic acid of any of claims 1046 to 1048, wherein the antigen-binding domain of the first CAR is a single-chain 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. [The present 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 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); 1046. The nucleic acid of the present invention, comprising: [This invention 1051] 1050. The nucleic acid of claim 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. [This invention 1052] The nucleic acid of the invention 1050 or 1051, wherein the antigen-binding domain of the first CAR is a single-chain 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. [This invention 1053] The nucleic acid of any of claims 1046 to 1053, 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. [This 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 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); The nucleic acid of any one of 1046 to 1053 of the present invention, comprising: [This invention 1055] 1054. The nucleic acid of claim 10, wherein the antigen-binding domain of the second 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:31. [The present invention 1056] The nucleic acid of 1054 or 1055, wherein the antigen-binding domain of the second CAR comprises 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. [This invention 1057] The nucleic acid of any of claims 1054 to 1056, wherein the antigen-binding domain of the second 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: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. [This invention 1058] The nucleic acid of any of claims 1054 to 1057, wherein the antigen-binding domain of the second CAR is a single-chain 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. [This invention 1059] The nucleic acid of any of claims 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. [The present invention 1060] 1059. The nucleic acid of any of claims 1046 to 1059, wherein the transmembrane domain of the first CAR and / or the second CAR is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, 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). [This invention 1061] The nucleic acid of any of claims 1046 to 1060, wherein the transmembrane domain of the first CAR and / or the second CAR comprises the transmembrane domain of CD8 alpha. [This invention 1062] The nucleic acid of any of claims 1046 to 1061, wherein the intracellular domain of the first CAR and / or the second CAR comprises a costimulatory signaling domain and an intracellular signaling domain. [This invention 1063] The nucleic acid of any of claims 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). [This invention 1064] The nucleic acid of any of claims 1046 to 1063, wherein the intracellular domain of the first CAR and / or the second CAR comprises the costimulatory domain of 4-1BB. [This invention 1065] The nucleic acid of any of claims 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 the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof. [The present invention 1066] The nucleic acid of any of claims 1046 to 1065, wherein the intracellular signaling domain of the first CAR and / or the second CAR comprises the intracellular domain of CD3ζ. [This 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; A nucleic acid comprising: 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) 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 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); Includes; 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 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); A nucleic acid comprising: [The present 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; A nucleic acid comprising: The first car is a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: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 Includes; The second car is a heavy chain variable region encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 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 A nucleic acid comprising: [The present 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; A nucleic acid comprising: the first CAR comprises a single-chain 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-chain 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. [The present 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; A nucleic acid comprising: the first polynucleotide sequence comprises a sequence 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 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; Nucleic acid. [This 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. [This invention 1072] 1071. The nucleic acid of the present invention, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [This invention 1073] The nucleic acid of the present invention 1071 or 1072, wherein 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. [This invention 1074] The nucleic acid of any one of 1071 to 1073 of the present invention, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody. [This 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. A nucleic acid comprising: [This invention 1076] 1075. The nucleic acid of the invention, wherein the second polynucleotide sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence encoding SEQ ID NO:53 or 54. [This invention 1077] The nucleic acid of claim 1075 or 1076, wherein the BiTE is capable of binding to wild-type EGFR (wtEGFR). [This invention 1078] The nucleic acid of claim 1075 or 1076, wherein the BiTE is capable of binding to EGFR variant III (EGFRvIII). [This invention 1079] The nucleic acid of any of claims 1046 to 1078, wherein the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker. [The present invention 1080] 1079. The nucleic acid of claim 1079, wherein the linker comprises a nucleotide sequence encoding an internal ribosome entry site (IRES) or a self-cleaving peptide. [This invention 1081] 1080. The nucleic acid of the present invention, wherein the self-cleaving peptide is a 2A peptide. [This invention 1082] 1081. The nucleic acid of the present invention, wherein the 2A peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), Equine rhinitis A virus 2A (E2A), and Foot-and-mouth disease virus 2A (F2A). [This invention 1083] The nucleic acid of claim 1081 or 1082, wherein the 2A peptide is T2A. [This invention 1084] The nucleic acid of any one of 1079 to 1083, wherein the linker further comprises a furin cleavage site. [This invention 1085] The nucleic acid of any one of 1079 to 1084 of the present invention, comprising, in the 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence. [The present invention 1086] The nucleic acid of any one of 1079 to 1084 of the present invention, comprising, in the 5' to 3' direction, a second polynucleotide sequence, a linker, and a first polynucleotide sequence. [This invention 1087] A vector comprising any one of the nucleic acids of the present inventions 1029 to 1086. [This invention 1088] The vector of the present invention 1087, which is an expression vector. [This invention 1089] The vector of the present invention 1087 or 1088, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector. [The present invention 1090] The vector of any one of 1087 to 1089 of the present invention, further comprising an EF-1a promoter. [This invention 1091] The vector of any one of 1087 to 1090, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [This invention 1092] The vector of any one of 1087 to 1091, further comprising a rev response element (RRE). [This invention 1093] The vector of any one of 1087 to 1092 of the present invention, further comprising a cPPT sequence. [This invention 1094] The vector of any one of 1087 to 1093 of the present invention, which is a self-inactivating vector. [This invention 1095] A modified immune cell or a precursor thereof, comprising a CAR of any one of the present inventions 1001 to 1027, a nucleic acid of any one of the present inventions 1029 to 1086, or a vector of any one of the present inventions 1087 to 1093. [This invention 1096] A modified immune cell or a precursor thereof comprising 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 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); 1. A modified immune cell or a precursor thereof, comprising: [This invention 1097] A modified immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20 1. A modified immune cell or a precursor thereof, comprising: [This invention 1098] 1. An engineered immune cell, or a precursor thereof, comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the CAR comprises a single-chain 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. [This invention 1099] 1. An engineered immune cell, or a precursor thereof, 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. [The present invention 1100] The modified cell of any of 1096 to 1099 of the present invention, wherein the CAR is capable of binding to IL13Rα2. [The present invention 1101] The modified cell of any of claims 1096 to 1100, wherein the CAR is capable of binding to human IL13Rα2. [The present invention 1102] The modified cell of any of 1096 to 1101 of the present inventions, further comprising an immune checkpoint inhibitor and secreting the immune checkpoint inhibitor. [The present invention 1103] The modified cell of the present invention 1102, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [The present invention 1104] The modified cell of claim 1102 or 1103, wherein 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. [This invention 1105] The modified cell of any of claims 1096 to 1104, further comprising an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, and secreting the BiTE. [The present invention 1106] 1105. The modified cell of the invention, 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. [This invention 1107] The modified cell of invention 1105 or 1106, wherein the BiTE is capable of binding to wild-type EGFR (wtEGFR). [This invention 1108] The modified cell of claim 1105 or 1106, wherein the BiTE is capable of binding to EGFR variant III (EGFRvIII). [This 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; 1. A modified immune cell or a precursor thereof, comprising: [The present invention 1110] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A modified immune cell or a precursor thereof comprising: 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) 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 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); Includes; 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 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); 1. A modified immune cell or a precursor thereof, comprising: [The present invention 1111] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A modified immune cell or a precursor thereof comprising: The first car is a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20 Includes; The second car is a heavy chain variable region comprising an amino acid sequence 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:32. 1. A modified immune cell or a precursor thereof, comprising: [The present invention 1112] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A modified immune cell or a precursor thereof comprising: the first CAR comprises a single-chain variable fragment (scFv) comprising an amino acid sequence 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-chain 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; Modified immune cells or their precursors. [The present invention 1113] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A modified immune cell or a precursor thereof comprising: the first CAR comprises an amino acid sequence 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; Modified immune cells or their precursors. [This invention 1114] The modified cell of any of 1109 to 1113 of the present inventions, further comprising an immune checkpoint inhibitor and secreting the immune checkpoint inhibitor. [This invention 1115] The modified cell of the present invention 1114, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [The present invention 1116] The modified cell of the present invention 1114 or 1115, wherein 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. [This invention 1115] The modified cell of any of 1096 to 1116 of the present invention, wherein the CAR is capable of binding to human IL13Rα2. [The present invention 1116] The second CAR targets wild-type EGFR (wtEGFR), mutant EGFR, and 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 The modified cell of any of claims 1109 to 1115, which is capable of binding to an EGFR isoform selected from the group consisting of EGFR variant II and EGFR variant II, or any combination thereof. [This invention 1117] The modified cell of any one of 1096 to 1116 of the present invention, which is a modified immune cell. [This invention 1118] The modified cell of any one of 1096 to 1117 of the present invention, which is a modified T cell. [This invention 1119] The modified cell of any one of 1096 to 1118 of the present invention, which is an autologous cell. [The present invention 1120] The modified cell of any of claims 1096 to 1119, which is an autologous cell obtained from a human subject. [This invention 1121] A pharmaceutical composition comprising a therapeutically effective amount of the modified cells of any one of the present inventions 1096 to 1120. [This invention 1122] A method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the modified cell of any of the present inventions 1096 to 1120, or the pharmaceutical composition of the present invention 1121. [This invention 1123] The method of claim 1122, wherein the disease is cancer. [This invention 1124] The method of claim 1123, wherein the cancer is glioma. [This invention 1125] The method of any one of claims 1123 to 1124, wherein the cancer is astrocytoma. [Invention 1126] The method of any one of claims 1123 to 1125, wherein the cancer is high-grade astrocytoma. [This invention 1127] The method of any one of claims 1123 to 1126, wherein the cancer is glioblastoma. [This invention 1128] 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising 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 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); A method comprising: [This invention 1129] 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, CAR, a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20 A method comprising: [The present invention 1130] 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising a chimeric antigen receptor (CAR) capable of binding to IL13Rα2, wherein the CAR comprises a single chain 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 SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:22. [This invention 1131] 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T 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:23 or SEQ ID NO:24 or SEQ ID NO:55 or SEQ ID NO:56. [This invention 1132] 1132. The method of any of claims 1128 to 1131, wherein the method further comprises the step of administering an immune checkpoint inhibitor, and wherein the modified cells secrete the immune checkpoint inhibitor. [This invention 1133] The method of claim 1132, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [This invention 1134] 1134. The method of any one of claims 1132 to 1133, wherein 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. [This invention 1135] The method of any of claims 1132 to 1134, wherein the immune checkpoint inhibitor is co-administered with the modified T cells. [This invention 1136] 1136. The method of any of claims 1128 to 1135, wherein the method further comprises administering an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, wherein the modified cells secrete the BiTE. [This invention 1137] The method of claim 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. [This invention 1138] The method of any one of claims 1136 to 1137, wherein the BiTE is capable of binding to wild-type EGFR (wtEGFR). [This invention 1139] The method of any one of claims 1136 to 1137, wherein the BiTE is capable of binding to EGFR variant III (EGFRvIII). [This invention 1140] The method of any of claims 1136 to 1139, wherein the BiTE is co-administered with the modified T cell. [This invention 1141] 1132. The method of any of claims 1128 to 1131, wherein the method further comprises administering an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, and an immune checkpoint inhibitor, wherein the modified cells secrete the BiTE and the immune checkpoint inhibitor. [This invention 1142] The method of claim 1141, wherein the BiTE and immune checkpoint inhibitor are co-administered with the modified T cells. [This invention 1143] 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 the epidermal growth factor receptor (EGFR) or an isoform thereof; 10. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising: [This invention 1144] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising: 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) 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 comprises the amino acid sequence TASLSVSSTYLH (SEQ ID NO:5) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); Includes; 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 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); A method comprising: [Invention 1145] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising: The first car is a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20 Includes; The second car is a heavy chain variable region comprising an amino acid sequence 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:32. A method comprising: [Invention 1146] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising: the first CAR comprises a single-chain variable fragment (scFv) comprising an amino acid sequence 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-chain 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; method. [This invention 1147] a first chimeric antigen receptor capable of binding to IL13Rα2; and A second chimeric antigen receptor (CAR) capable of binding to the epidermal growth factor receptor (EGFR) or its isoforms 1. A method of treating glioblastoma in a subject in need thereof, comprising administering to the subject an effective amount of modified T cells comprising: the first CAR comprises an amino acid sequence 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; method. [Invention 1148] The method of any of claims 1143 to 1147, wherein the method further comprises the step of administering an immune checkpoint inhibitor, and wherein the modified cells secrete the immune checkpoint inhibitor. [This invention 1149] 1148. The method of claim 1148, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, and TIM-3. [This invention 1150] 1149. The method of any one of claims 1148 to 1149, wherein 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. [This invention 1151] The method of any of claims 1148 to 1150, wherein the immune checkpoint inhibitor is co-administered with the modified T cells. [This invention 1152] The nucleic acid of any of claims 1028 to 1086, wherein the nucleic acid further comprises an inducible promoter, and the inducible promoter comprises a nucleotide sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 161, 162, or 198. [This invention 1153] A nucleic acid comprising a polynucleotide sequence encoding a CAR comprising 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. [This invention 1154] 1153. The nucleic acid of the invention, wherein the linker comprises 5, 10, 15, or 20 amino acids. [This invention 1155] 1153. The nucleic acid of the invention, 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 an isoform thereof. [Invention 1156] 1155. The nucleic acid of the present invention, wherein the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 163, 165, 167, or 169, and / or is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 164, 166, 168, or 170. [This invention 1157] A nucleic acid comprising a polynucleotide sequence encoding parallel CARs, wherein the parallel CARs comprise a first CAR and a second CAR, each comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the first CAR and second CAR are separated by a cleavable linker. [This 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. [This invention 1159] A nucleic acid comprising a polynucleotide sequence encoding a BiTE and a CAR. [The present invention 1160] 1159. The nucleic acid of the invention, wherein the BiTE comprises an antigen-binding domain capable of binding to EGFR or an isoform thereof, and the CAR comprises an antigen-binding domain capable of binding to IL13Rα2. [This invention 1161] 1159. The nucleic acid of the invention, wherein the BiTE comprises an antigen-binding domain capable of binding to IL13Rα2, and the CAR comprises an antigen-binding domain capable of binding to EGFR or an isoform thereof. [This invention 1162] A nucleic acid of the 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. [This invention 1163] A nucleic acid of the 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 comprising a polynucleotide sequence encoding a first BiTE and a second BiTE. [This invention 1165] The nucleic acid of claim 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 an isoform thereof. [Invention 1166] 1164. A nucleic acid of the invention, wherein the polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:180. [This invention 1167] A nucleic acid of the 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 drawings]

[0099] The foregoing and other features and advantages of the present invention will be better understood from the following detailed description of illustrative embodiments taken in conjunction 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 by human T cells after electroporation of mRNA for mouse scFv- and humanized scFv (07 and 08)-based CAR constructs using rabbit anti-mouse 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 coculture 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 cocultured with the human tumor cell line in Figure 1D, using untransduced T cells (UTD) as a control. Human CD8 was stained to distinguish CD4- and CD8-positive T cell subpopulations along the x-axis. Figure 1F shows results from a chromium release assay of humanized IL13Rα2 CAR T cells cocultured with the tumor cell line in Figure 1D at different effector / target (E:T) ratios (1:1, 3:1, 10:1, and 30:1) compared with untransduced T cells (UTD) using a one-way analysis of variance (ANOVA) post hoc Tukey's test. **P<0.01, ***P<0.001, ****P<0.0001. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 2A]Figures 2A-2E illustrate the finding that IL13Rα2 CAR T cells control tumor growth in vivo. Figure 2A shows flow-based EGFRvIII and IL13Rα2 expression on the D270 tumor cell line using a control antibody. Figure 2B illustrates EGFRvIII-targeted (2173BBz) and IL13Rα2-targeted (Hu08BBz) CAR T cells cocultured with the D270 tumor cell line. T cell stimulation was demonstrated by staining with FITC-conjugated anti-CD69 antibody, and median fluorescence intensity (MFI) was quantified for CD4 and CD8 CAR-positive T cells after 24 or 48 hours of coculture using untransduced T cells as a control. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey's test. Figure 2C illustrates human T cells counted in the spleens of D270-injected NSG mice (n = 3) 11 days after the intravenous transfer of equal numbers of untransduced T cells, EGFRvIII-targeted (2173BBz) CAR T cells, or IL13Rα2-targeted (Hu08BBz) CAR T cells. Figure 2D illustrates the intravenous 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 untransduced T cells, 7 days after tumor implantation in an NSG mouse model (n = 10 / group) implanted subcutaneously with D270. Tumor volume measurements (left panel) and bioluminescence imaging (middle panel) were performed to assess tumor growth. Linear regression was used to test for significant differences between experimental groups. Predefined IACUC-approved morbidity endpoints were defined as mice hunch, becoming unable to walk, or tumors reaching 2 cm in any direction. Kaplan-Meier curves were used to plot survival based on time to endpoint (Prism software). Statistical significance was determined using the log-rank test.Figure 2E illustrates that 800,000 IL13Rα2-targeting CAR-positive (Hu08BBz) CAR T cells or the same number of non-transduced T cells were given intravenously 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 described in Figure 2D. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 3A]Figures 3A-3D illustrate the finding that checkpoint blockade selectively enhances CAR T cell function. Figure 3A illustrates that EGFRvIII (2173BBz)-targeted CAR T cells and IL13Rα2 (Hu08BBz)-targeted CAR T cells, as well as untransduced T cell controls, were cocultured with the target-positive D270 tumor cell line and the target-negative A549 tumor cell line. Checkpoint receptor expression on T cells was determined by flow cytometry and staining with fluorochrome-conjugated anti-checkpoint receptor antibodies; median fluorescence intensity (MFI) was quantified for CD4 CAR-positive and CD8 CAR-positive T cells after 24 or 48 hours of coculture. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey's test. Figure 3B illustrates that untransduced (UTD) human T cells were intravenously injected into a mouse model (n = 5 / group) subcutaneously implanted with D270 tumor cells 7 days after tumor implantation. Starting on day 6, 200 μg of PBS or the same volume of checkpoint blockade antibodies (anti-PD-1, anti-CTLA-4, and anti-TIM-3) was intraperitoneally injected every four 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) 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 group was compared with the CAR T cell control group combined with PBS (n = 5 / group). Figure 3D illustrates a comparison of different checkpoint blockade combination therapies based on tumor size in mice in the EGFRvIII-targeted (2173BBz) and IL13Rα2-targeted (Hu08BBz) CAR T cell groups. Survival curves were also compared between these two CAR T cell groups. Statistical significance of differences in tumor growth between experimental groups was determined by linear regression, and the log-rank test was used to determine statistical significance of differences in survival curves. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to 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 shows a size-based vector map of each component of the minibody-secreted anti-IL13Rα2 CAR design. The minibody is simplified as a PD-1-targeting scFv, a CTLA-4-targeting scFv, and a TIM-3-targeting scFv linked to a human IgG1 spacer and CH3 domain. A self-cleaving sequence (P2A) was used to express the minibody together with the anti-IL13Rα2 CAR in the same open reading frame. Figure 4B illustrates that CAR expression was detected not only on minibody-secreting IL13Rα2-targeting CAR T cells but also on non-minibody-secreting IL13Rα2-targeting CAR T cells. Figure 4C illustrates that supernatants from anti-PD-1 minibody-secreting IL13Rα2-targeted CAR T cells and anti-CTLA-4 minibody-secreting IL13Rα2-targeted CAR T cells were collected and concentrated separately. 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 using an unpaired t-test. Figure 4D illustrates that untransduced T cells, IL13Rα2-targeted (Hu08BBz) CAR T cells, and minibody-secreting Hu08BBz CAR T cells were cocultured with the D270 tumor cell line. Median fluorescence intensity (MFI) was quantified by BV605-conjugated anti-TIM-3 antibody staining of the CD4 and CD8 subpopulations of CAR-positive T cells after 24 or 48 hours of coculture. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey's test. Figure 4E illustrates that 8 days after subcutaneous implantation of D270 (n=8), 800,000 IL13Rα2-targeting (Hu08BBz) CAR T cells and minibody-secreting Hu08BBz CAR T cells or the same number of untransduced T cells were intravenously injected. Tumor size was measured with a caliper and compared between each group. 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] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5A] Figures 5A-5D illustrate the finding that IL13Rα2 CAR T cells respond to canine tumors. Figure 5A shows IL13Rα2 expression analysis for 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) electroporated with mRNA. Intracellular cytokine (IFNγ) staining was performed after co-culture of these CAR T cells with human and canine IL13Rα2 proteins, using bovine serum albumin (BSA) as a control. CD8 staining was used to distinguish CD4-positive and CD8-positive T cell populations on the x-axis. Figure 5C illustrates the expression of canine IL13Rα1 mRNA and IL13Rα2 mRNA for various canine tumor cell lines (Camac2, CLBL-1, GL-1, Cacal3, Cacal5, BW-KOSA, CS-KOSA, MC-KOSA, and SK-KOSA) detected by reverse transcription-polymerase chain reaction (RT-PCR) using canine GAPDH as a control. After co-culture of mRNA-electroporated IL13Rα2-targeted (Hu07BBz and Hu08BBz) human CAR T cells and untransduced T cells with the aforementioned canine tumor cell lines, the percentages of cytokine (IFNγ, IL2, and TNFα)-positive T cells within CD4+ and CD8+ T cell subpopulations were analyzed. Figure 5D illustrates that 5 million MC-KOSA cells were subcutaneously implanted (n=5 / group) 7 days later, 2 million Hu08BBz-transduced human CAR-positive T cells were injected intravenously. Tumor size was measured with calipers and compared with a control group of the same amount of non-transduced T cells. Statistical significance of tumor growth was determined by linear regression. ****P<0.0001. [Figure 5B]See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6E illustrate the finding that canine IL13Rα2 CAR T cells control canine tumor growth. Figure 6A illustrates that mRNA-electroporated Hu08BBz canine CAR T cells were cocultured 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 with non-transduced canine T cells. Figure 6B shows vector maps of the anti-IL13Rα2 human Hu08BBz CAR construct (Hu08HuBBz) and canine Hu08BBz CAR construct (Hu08CaBBz). Figure 6C illustrates the co-culture of mRNA-electroporated Hu07HuBBz, Hu08HuBBz, and Hu08CaBBz canine CAR T cells with CLBL1 and J3T tumor cell lines. Canine IFNγ secretion was detected by ELISA. An unpaired t-test was used to determine the statistical significance of IFNγ secretion between Hu08HuBBz and Hu08CaBBz co-cultured with J3T glioma cells. Figure 6D illustrates the J3T canine glioma cell line orthotopically implanted into the brain of NSG mice. At 7, 10, and 13 days after tumor implantation, 12 million electroporated Hu08HuBBz, Hu08CaBBz, or non-transduced canine T cells were intravenously injected into the mouse model (n = 4 / group). Tumor growth was assessed every 3–4 days by bioluminescence imaging. Statistical significance of tumor growth was determined by linear regression. Figure 6E illustrates that canine T cells used in the second injection on day 10 were analyzed for CAR expression and canine IFNγ secretion after co-culture with the J3T tumor cell line. Canine CD4 was stained to distinguish canine CD4- and CD8-positive subpopulations along the x-axis. ns, not significant; **P<0.01, ****P<0.0001. [Figure 6B] See legend to Figure 6A. [Figure 6C]See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7A] Figures 7A-7C illustrate IL13Rα1 and IL13Rα2 expression panels in human normal or tumor tissues. Figures 7A-7B show IL13Rα1 and IL13Rα2 expression in human normal tissues reported as mean TPM (transcripts parts per million) based on RNA-seq data from the Human Protein Atlas (HPA) (www.proteinatlas.org). Figure 7C shows IL13Rα2 expression in human tumors, reported as median expression based on The Cancer Genome Atlas (TCGA) data available at cBioPortal. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8A] Figures 8A-8D illustrate mouse scFv-based IL13Rα2-targeted CAR T cells. Figure 8A shows the vector map of the mouse scFv-based anti-IL13Rα2 CAR design tested. Figure 8B illustrates the expression of mouse scFv (07 and 08)-based IL13Rα2-targeted CAR constructs on electroporated human T cells. Figure 8C illustrates IL13Rα1 and IL13Rα2 expression analysis for 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) cocultured with the human tumor cell lines in Figure 8C, as compared to untransduced T cells (UTD). Human CD8 was stained to distinguish CD4- and CD8-positive subpopulations of T cells along the x-axis. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A] Figures 9A-9C illustrate humanized IL13Rα2-targeted CAR T cells cocultured with human normal cell types. Figure 9A illustrates flow-based CAR expression staining of humanized IL13Rα2 CAR-transduced T cells used in the coculture experiments. Figure 9B illustrates flow cytometry analysis of IL13Rα1 and IL13Rα2 expression on human normal cells (CD34-positive bone marrow 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 cocultured with human normal cells in Figure 9B, using untransduced T cells (UTD) as a control. Human CD3 and CD8 were stained to distinguish CD4- and CD8-positive subpopulations of T cells along the x-axis. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10-1] Figures 10A-10E illustrate the stimulation and expansion of IL13Rα2-targeted CAR T cells cocultured in vitro. Figures 10A-10C illustrate flow-based intracellular cytokine (IFNγ, IL2, and TNFα) staining of murine IL13Rα2 CAR T cells cocultured with a human tumor cell line (Figure 10A), humanized IL13Rα2 CAR T cells cocultured with a human tumor cell line (Figure 10B), and humanized IL13Rα2 CAR T cells cocultured with human normal cells (Figure 10C). The percentages of cytokine-positive T cells within the CD4-positive and CD8-positive subpopulations are shown. Figure 10D illustrates flow-based EGFRvIII and IL13Rα2 expression for the D270 tumor cell line at days 0, 1, 2, 3, 5, and 7 of in vitro culture using a control antibody. Figure 10E illustrates a T cell proliferation assay determined by flow cytometry by CFSE staining performed on UTD T cells, 2173BBz CAR-positive T cells, and Hu08BBz CAR-positive T cells at days 3, 5, and 8 co-cultured with D270 cell line, with A549 cell line as a control. [Figure 10-2] See description of Figure 10-1. [Figure 11A] Figures 11A-11B illustrate surface marker staining for CAR T cells cocultured 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 cocultured with the D270 cell line for 48 hours. Live CD45+, CD3+ lymphocytes were gated, and T cell surface marker expression 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+ and CD8+ T cells determined by flow cytometry after 24 or 48 hours of coculture by staining with the corresponding antibodies conjugated with fluorescent dyes. Representative expression results are shown for UTD T cells and CAR+ T cells cocultured with the D270 cell line. [Figure 11B] See legend to 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 and CD8-positive T cell subpopulations on days 0, 3, 7, and 13 during in vitro expansion of T cells using anti-CD3 and anti-CD28 beads. Figure 12B illustrates flow-based detection of expression analysis of checkpoint receptor ligands (PD-L1, CD80, CD86, and Galectin-9) for the D270 glioma cell line. Figure 12C illustrates human PD-1, CD69, CD4, and CD8 staining on human CD3+ T cells in ex vivo mouse spleens after infusion of 2173BBz CAR T cells in combination with anti-PD-1 checkpoint blockade in a D270 subcutaneously implanted NSG mouse model. Data are shown as the percentage of positive cells. Statistical significance was calculated by unpaired t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13]Figures 13A-13C illustrate the in vitro analysis of minibody-secreting T cells (MiST) cocultured with target cells. Figure 13A illustrates the coculture of untransduced T cells, IL13Rα2-targeted (Hu08BBz) CAR T cells, and minibody-secreting Hu08BBz CAR T cells (anti-PD1 and anti-CTLA4 MiST) with the D270 tumor cell line. Median fluorescence intensity (MFI) was quantified by staining with BV711-conjugated anti-PD1 and PE-conjugated anti-CTLA-4 antibodies for the CD4 and CD8 subpopulations of CAR-positive T cells after 24 or 48 hours of coculture. Figure 13B illustrates the stimulation of IL13Rα2 (Hu08BBz)-targeted CAR T cells and minibody-secreting cells assessed after coculture with the D270 tumor cell line. Median fluorescence intensity (MFI) was quantified by FITC-conjugated anti-CD69 antibody staining on the CD4 and CD8 subpopulations of CAR-positive T cells after 24 or 48 hours of coculture. Figure 13C illustrates that the percentage of cytokine (IFNγ, IL2, and TNFα) staining-positive T cells within the CD4- and CD8-positive T cell subpopulations was analyzed for IL13Rα2-targeted (Hu08BBz) CAR T cells and minibody-secreting cells after coculture with the D270 target tumor cell line. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey's 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 sequence of IL13Rα2 and a canine osteosarcoma mouse model. Figure 14A illustrates a comparison of the amino acid sequences of human and canine IL13Rα2 using Geneious software. Figure 14B illustrates that canine osteosarcoma tumor cell lines (BW-KOSA, CS-KOSA, MC-KOSA, and SK-KOSA) were subcutaneously implanted at different doses into the right flank of NSG mice. Bioluminescence imaging was performed repeatedly to assess tumor growth in each group. [Figure 14B] See legend to Figure 14A. [Figure 15]Figures 15A-15B illustrate the nucleotide sequences of inducible promoters disclosed herein. Figure 15A: DNA sequence for an inducible promoter that can drive expression after T cell activation. Portions of this sequence can be repeated to enhance expression levels in T cells. This promoter can engineer T cells / CAR T cells to express a designed RNA or amino acid. Figure 15B: The sequence (SEQ ID NO:198) repeated to enhance activity is underlined. [Figure 16] Figures 16A-16B illustrate the functional activity of an inducible promoter. Figure 16A is a schematic diagram of an inducible promoter-containing construct, including the TDTomato gene for fluorescent expression. Figure 16B shows the expression of TD-Tomato in Jurkat cells (a 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-18E show the amino acid and nucleic acid sequences for a tandem CAR with a 5 AA linker ((G4S); Figure 18A), a tandem CAR with a 10 AA linker (2(G4S); Figure 18B), a tandem CAR with a 15 AA linker (3(G4S); Figure 18C), a tandem CAR with a 20 AA linker (4(G4S); Figure 18D), and a parallel CAR (Figure 18E). [Figure 18B]See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 19] Quantitation of CAR construct expression as determined by flow cytometry is shown. 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-conjugated PE or streptavidin-conjugated PE alone. [Figure 20] Figure 1 illustrates the stimulation of T cells containing Hu08BBz CAR and 806BBz CAR, Hu08 / 806 bispecific CAR, and Hu08BBz_P2A_806BBz parallel CAR. Each CAR T cell population was co-cultured with the 5077 glioma stem cell line overexpressing the target. CAR1 (Hu08BBz) and CAR2 (806BBz) were single CAR constructs, while 5AA, 10AA, 15AA, and 20AA were tandem bispecific CAR constructs of various lengths (Hu08 / 806_(G4S), Hu08 / 806_2(G4S), Hu08 / 806_3(G4S), Hu08 / 806_4(G4S)). 2A was a parallel bispecific CAR construct (Hu08BBz_P2A_806BBz). T cell stimulation was illustrated by staining with APC-conjugated anti-CD69 antibody, and median fluorescence intensity (MFI) was quantified for CD4+ ( FIG. 20 , top) and CD8+ ( FIG. 20 , bottom) CAR-positive T cells after 24 hours of co-culture, with non-transduced T cells as a control. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey's 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 [IFNγ (Figures 21A and 21D), IL2 (Figures 21B and 21E), and TNFα (Figures 21C and 21F)] staining of each of the tandem bispecific CAR T cells and parallel CAR T cells from Figures 18-20 co-cultured with the target-overexpressing 5077 glioma stem cell line. The percentage of cytokine-positive T cells within the CD4+ (Figures 21A-21C) and CD8+ (Figures 21D-21F) T cell subpopulations was demonstrated. One-way ANOVA post hoc Tukey's test. **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Figure 21-2] See description of 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 not expressing EGFRvIII and IL13Rα2 (5077_Rα2-_vIII-), or overexpressing IL13Rα2 alone (5077_Rα2+_vIII-), overexpressing EGFRvIII alone (5077_Rα2-_vIII+), or overexpressing EGFRvIII and IL13Rα2 (5077_Rα2+_vIII+), as well as control untransduced T cells (UTD). Figure 2A illustrates the bioluminescence-based cytotoxicity assay of the 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 a 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 a 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 a bioluminescence-based cytotoxicity assay of the Hu08 / 806_4(G4S) bispecific CAR. The linker between the two scFvs is GGGGSx4 (SEQ ID NO: 160). Data are presented as mean±SEM. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 23]Figures 23A-23D illustrate the in vitro killing of a parallel bispecific CAR construct (Hu08BBz_P2A_806BBz). Bioluminescence-based cytotoxicity assays were performed to test the killing ability of 806BBz / Hu08BBz (Hu08BBz_P2A_806BBz) parallel bispecific CAR T cells when cocultured with target 5077 cell lines overexpressing IL13Rα2 alone (5077_Rα2+_vIII-), EGFRvIII alone (5077_Rα2-_vIII+), or EGFRvIII and IL13Rα2 (5077_Rα2+_vIII+) and D270 glioma cell lines overexpressing EGFRvIII and IL13Rα2 (D270_Rα2+_vIII+), as well as control untransduced T cells (UTD). Data are presented as mean ± SEM. [Figure 24] This figure illustrates that 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 806BBz / Hu08BBz bispecific CAR T cells or the same number of untransduced T cells (UTD). Tumor volume measurements (Figure 24, top) were performed to assess tumor growth. Linear regression was used to test for significant differences between experimental groups. Predefined IACUC-approved morbidity endpoints were defined as mice hunched over, unable to walk, or tumors reaching 2 cm in any direction. Kaplan-Meier curves were used to plot survival based on time to endpoint (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 anti-IL13Rα2 / CD3 bispecific T cell engager (Hu07BiTE) in IL13Rα2-positive cells. Fresh medium (Figure 25A) and 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 in coculture with the 5077 cell line (top, IL13Rα2-) or 4892 cell line (bottom, IL13Rα2+). CD69 staining was used to demonstrate T cell activation. Human CD8 staining was used to distinguish CD4- and CD8-positive subpopulations of T cells along the x-axis. [Figure 26] Figure 1 illustrates the in vitro binding of the anti-IL13Rα2 / CD3 (Hu08OKT3) bispecific T cell engager to IL13Rα2. 293T cells were transfected with the plasmid pTRPE CFP (fluorescent gene) or pTRPE Hu08BiTE. Supernatants were collected after 2 days. Direct ELISA was performed to detect binding of Hu08OKT3 BiTE to the recombinant protein IL13Rα2. [Figure 27] Figure 1 illustrates the in vitro binding of two anti-EGFR / CD3 (C225BiTE and 806BiTE) bispecific T cell engagers to EGFR. T cells were transduced with pTRPE Hu08BBz, pTRPE C225BiTE, or pTRPE 806BiTE, with untransduced T cells (UTD) and Hu8BBz CAR serving as controls. Supernatants were collected after 7 days. Direct ELISA was performed to detect binding of BiTEs to the recombinant proteins, EGFR wild-type, or EGFRvIII. [Figure 28A]Figures 28A-28B illustrate the differential 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 not IL13Rα2. 806BiTE- and C225BiTE-transduced T cells were cocultured with wild-type or EGFRvIII-overexpressing 5077 cells, and a killing assay (Figure 28A) and cytokine secretion assay (Figure 28B) were performed. Figure 28A illustrates that 806BiTE-transduced T cells killed only EGFRvIII-overexpressing 5077 cells, whereas C225BiTE-transduced T cells killed both wild-type and EGFRvIII-overexpressing 5077 cells. Figure 28B illustrates that 806BiTE induced secretion of INFγ, IL-2, and TNF only when 806BiTE-transduced T cells were cocultured with EGFRvIII-overexpressing 5077 cells, whereas C225BiTE-transduced T cells stimulated secretion of INFγ, IL-2, and TNF in the absence and presence of EGFRvIII variants. No cytokine production was observed in the absence of target cells. [Figure 28B] See legend to Figure 28A. [Figure 29] This figure illustrates T cell activation induced by anti-IL13Rα2 / CD3 (Hu08 / KT3-T2A-mCherry) and anti-EGFRvIII / CD3 (80 / KT3-T2A-mCherry) bispecific T cell engagers in IL13Rα2- and EGFRvIII-positive cells. Supernatants from untransduced T cells (UTD), 806BBz CAR T cells, 806BiTE T cells, Hu08BBz CAR T cells, and Hu08BiTE T cells were collected and used to coculture untransduced T cells with target-overexpressing 5077 GSC line and D270 glioma cell line. CD69 staining was used to demonstrate T cell activation. Human CD8 staining was used to distinguish CD4- and CD8-positive subpopulations of T cells along the x-axis. [Figure 30]Figures 30A-30D illustrate schematic diagrams of the bispecific constructs used in the BiTE / CAR experiments. Figure 30A shows a schematic diagram of a parallel bispecific polynucleotide sequence (806BBz / Hu08BBz bispecific construct) comprising 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) comprising 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) comprising a first nucleotide sequence encoding an anti-IL13Rα2 / CD3 bispecific T cell engager, a second nucleotide encoding an 806CAR, and a third nucleotide encoding a fluorescent marker. Figure 30D shows a schematic polynucleotide sequence (806BiTE / Hu08BiTE bispecific construct) comprising a first nucleotide sequence encoding an anti-EGFRvIII / CD3 bispecific T cell engager, a second nucleotide encoding an anti-IL13Rα2 / CD3 bispecific T cell engager, and a third nucleotide encoding a fluorescent marker. Self-cleaving sequences (P2A and / or T2A) link the CAR, bispecific T cell engager, and fluorescent marker in the same open reading frame. [Figure 31A] Figures 31A-31D show the amino acid and nucleic acid sequences for 806BBz / Hu08BBz set forth as SEQ ID Nos: 173-174 (Figure 31A), 806BiTE / Hu08BBz set forth as SEQ ID Nos: 175-176 (Figure 31B), Hu08BiTE / 806BBz set forth as SEQ ID Nos: 177-178 (Figure 31C), and 806BiTE / Hu08BiTE set forth as SEQ ID Nos: 179-180 (Figure 31D). [Figure 31B] See legend to Figure 31A. [Figure 31C] See legend to Figure 31A. [Figure 31D] See legend to 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 cocultured with target-overexpressing (EGFRvIII / IL13Rα2) cell lines, with untransduced T cells (UTD) as a control. Data are presented as mean ± SEM. Figure 32A shows the cytotoxic effect in a 5077 cell line overexpressing EGFRvIII alone (5077_Rα2-_vIII+). Figure 32B shows the cytotoxic effect in a 5077 cell line overexpressing IL13Rα2 alone (5077_Rα2+_vIII-). Figure 32C shows the cytotoxic effect in a 5077 cell line overexpressing IL13Rα2 and EGFRvIII (5077_Rα2+_vIII+). FIG. 32D shows the cytotoxic effect in the D270 cell line overexpressing IL13Rα2 and EGFRvIII (D270_Rα2+_vIII+). [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 (UTD) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 33A shows reduced tumor size in animals treated with 806BiTE / Hu08BBz bispecific T cells. Tumor volume measurements were performed to assess tumor growth. Linear regression was used to test for significant differences between experimental groups. Predefined IACUC-approved morbidity endpoints were defined as mice hunching over, becoming unable to walk, or tumors reaching 2 cm in any direction. Figure 33B shows improved survival in animals treated with 806BiTE / Hu08BBz bispecific T cells. Kaplan-Meier curves were used to plot survival based on time to endpoint (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 potential of Hu08BiTE / 806BBz bispecific T cells when cocultured with target-overexpressing (EGFRvIII / IL13Rα2) 5077 and D270 glioma cell lines, with untransduced T cells (UTD) as a control. Data are presented as mean ± SEM. Figure 34A shows the cytotoxic effect in a 5077 cell line overexpressing EGFRvIII alone (5077_Rα2-_vIII+). Figure 34B shows the cytotoxic effect in a 5077 cell line overexpressing IL13Rα2 alone (5077_Rα2+_vIII-). Figure 34C shows the cytotoxic effect in a 5077 cell line overexpressing IL13Rα2 and EGFRvIII (5077_Rα2+_vIII+). Figure 34D shows the cytotoxic effect in the D270 cell line overexpressing IL13Rα2 and EGFRvIII (D270_Rα2+_vIII+). [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 (UTD) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 35A shows reduced tumor size in animals treated with Hu08BiTE / 806BBz bispecific T cells. Tumor volume measurements were performed to assess tumor growth. Linear regression was used to test for significant differences between experimental groups. Predefined IACUC-approved morbidity endpoints were defined as mice hunching over, becoming unable to walk, or tumors reaching 2 cm in any direction. Figure 35B shows improved survival in animals treated with Hu08BiTE / 806BBz bispecific T cells. Kaplan-Meier curves were used to plot survival based on time to endpoint (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 cocultured with target-overexpressing (EGFRvIII / IL13Rα2) 5077 and D270 glioma cell lines, with untransduced T cells (UTD) as a control. Data are presented as mean ± SEM. Figure 36A shows the cytotoxic effect in the 5077 cell line overexpressing EGFRvIII alone (5077_Rα2-_vIII+). Figure 36B shows the cytotoxic effect in the 5077 cell line overexpressing IL13Rα2 alone (5077_Rα2+_vIII-). Figure 36C shows the cytotoxic effect in the 5077 cell line overexpressing IL13Rα2 and EGFRvIII (5077_Rα2+_vIII+). Figure 36D shows the cytotoxic effect in the D270 cell line overexpressing IL13Rα2 and EGFRvIII (D270_Rα2+_vIII+). [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 (UTD) were intravenously injected into NSG mice (n=8 / group) subcutaneously implanted with D270. Figure 37A shows reduced tumor size in animals treated with 806BiTE / Hu08BiTE bispecific T cells. Tumor volume measurements were performed to assess tumor growth. Linear regression was used to test for significant differences between experimental groups. Predefined IACUC-approved morbidity endpoints were defined as mice hunching over, becoming unable to walk, or tumors reaching 2 cm in any direction. Figure 37B shows improved survival in animals treated with 806BiTE / Hu08BiTE bispecific T cells. Kaplan-Meier curves were used to plot survival based on time to endpoint (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 intratumoral injection, we illustrate the diffusion of therapeutic agents into the contralateral ventricle following intraventricular injection into the other ventricle. 5 μL of trypan blue was injected into the right ventricle 1-2 mm right of bregma, 0.3 mm anterior, and 3.0 mm deep. Within 15 minutes of injection, the animals were euthanized, and the brains were examined for diffusion of trypan blue into the contralateral ventricle. Blue staining in both ventricles indicates both successful injection of therapeutic agents into the right ventricle and successful diffusion of therapeutic agents into the left contralateral ventricle. DETAILED DESCRIPTION OF THE INVENTION

[0101] Detailed Description The present invention provides compositions and methods for engineered immune cells or their precursor cells (e.g., engineered 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 engineered immune cells or their precursor 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 isoforms. The provided compositions and methods are useful for treating cancer (e.g., glioma, high-grade astrocytoma, and glioblastoma).

[0102] It is understood that the methods described in this disclosure are not limited to the particular methods and / or experimental conditions disclosed herein, as such methods and conditions may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0103] Furthermore, unless otherwise indicated, the experiments described herein use conventional molecular cell biology and immunological techniques within the skill of those skilled in the art.Such techniques are well known to skilled workers and are fully described in literature.For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, MR Green and J. Sambrook's Molecular Cloning: A Laboratory Manual (Fourth Edition) 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 defined, scientific and technical terms used herein have meanings that are commonly understood by those skilled in the art. In the event of any potential semantic ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or." The use of the term "including" and other forms such as "includes" and "included" is non-limiting.

[0105] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and widely used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art, unless otherwise specified, as described in various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are generally performed according to manufacturer's specifications as described herein or as commonly accomplished in the art. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0106] In order that this disclosure may be more readily understood, selected terms are defined below.

[0107] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0108] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0109] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" particularly refers to T cells undergoing cell division.

[0110] As used herein, "alleviating" 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 provokes an immune response. This immune response may include either or both antibody production or activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule can serve as an antigen, including any protein or peptide.

[0112] Furthermore, antigens can be derived from recombinant 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 therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0113] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into that individual.

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

[0115] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0116] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate unless the disease is ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be without the disorder. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

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

[0118] "Effective amount" or "therapeutically effective amount," as used interchangeably herein, refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. Immune responses can be readily assessed by numerous art-recognized methods. Those skilled in the art will understand that the amount of a composition administered herein will vary and can be readily determined based on numerous factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered.

[0119] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0120] As used herein, "endogenous" refers to any material that originates 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 elicit an immune response and induce a B cell response and / or a T cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. Generally, epitopes are approximately 10 amino acids and / or sugars in size. Preferably, epitopes are about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12 amino acids, and most preferably about 8-10 amino acids. Those skilled in the art will understand that, in general, the overall three-dimensional structure of a molecule, rather than its specific linear sequence, is the primary criterion for antigen specificity, and therefore, this distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0122] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0123] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number relative to the number originally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

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

[0125] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those 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 a recombinant polynucleotide.

[0126] As used herein, "identity" refers to the identity of the subunit sequence between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, 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 an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, 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 that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

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

[0129] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0130] " Lentivirus " as used herein refers to a genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, so they are one of the most efficient gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0131] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell 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" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0133] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0134] The term "oligonucleotide" typically refers to a short polynucleotide. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it should be understood that it also includes an RNA sequence in which "T" is replaced by "U" (i.e., A, U, C, G).

[0135] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that a nucleotide sequence encoding a protein, depending on its form, may contain introns.

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

[0137] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into 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 nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0138] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0139] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a 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 cross-species 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 allelic forms 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 binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction 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 the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0141] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0142] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, 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) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include livestock and pets, such as, for example, ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0144] "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, a 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 term "T cell receptor" or "TCR" refers to a membrane protein complex involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of an alpha (α) chain and a beta (β) chain, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain, and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains 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 prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0147] "Graft" refers to a biocompatible lattice or donor tissue, organ, or cells to be transplanted. Examples of grafts include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Grafts can also refer to any material to be administered to a host. For example, grafts can refer to nucleic acids or proteins.

[0148] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0149] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0150] A "vector" is a material composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into 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. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0151] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth 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 engineered immune cells or their precursor cells, such as engineered T cells, containing the CAR are also provided. Thus, in some embodiments, the immune cells are genetically engineered to express the CAR. Nucleic acids encoding the CAR, vectors encoding the nucleic acids, and engineered cells (e.g., engineered T cells) containing the CAR, vector, or nucleic acid are also provided.

[0153] A subject CAR of the present invention comprises an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, and an intracellular domain. A subject CAR of the present invention may optionally comprise a hinge domain. Thus, a subject 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 can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain (both described elsewhere herein) for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, and is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0155] The antigen binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CAR of the present invention.The subject CAR of the present invention can also include the hinge domain described herein.The subject CAR of the present invention can also include the spacer domain described herein.In some embodiments, each of the antigen binding domain, transmembrane domain, and intracellular domain is separated by a linker.

[0156] In certain embodiments, the CAR is capable of binding to human IL13Rα2. In certain embodiments, the CAR is capable of binding to canine IL13Rα2. In certain embodiments, the CAR is capable of binding to canine IL13Rα2 and human IL13Rα2.

[0157] In one aspect, the present invention provides 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), where 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), 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).

[0158] In another aspect, the present invention includes 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), 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).

[0159] Tolerated variations in the CAR sequence will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, or 18.

[0160] In another aspect, the present invention includes 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 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 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 includes 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 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 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, the CAR is capable of binding to GBM stem cells.

[0165] antigen-binding domain The antigen-binding domain of a CAR is the extracellular region of the CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. In certain embodiments, the antigen-binding domain is capable of binding to IL13Rα2. In certain embodiments, the antigen-binding domain is capable of binding to human IL13Rα2. In certain embodiments, the antigen-binding domain is capable of binding to canine IL13Rα2. In certain embodiments, the antigen-binding domain is capable of binding to human IL13Rα2 and canine IL13Rα2.

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

[0167] In certain embodiments, 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 certain embodiments, 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 certain embodiments, 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 certain embodiments, 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 antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. In certain embodiments, the antigen-binding domain comprises an scFv capable of binding to IL13Rα2. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:10. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:11. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:21. In certain embodiments, the antigen-binding domain comprises 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 antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. In certain embodiments, the antigen-binding domain comprises an scFv capable of binding to IL13Rα2. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 125. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 127. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 129. In certain embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 131.

[0173] Tolerated variations in antigen-binding domain sequences will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0174] Tolerated variations in antigen-binding domain sequences will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0175] Tolerated variations in antigen-binding domain sequences will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NOs: 2, 77, 79, 82, 84, 86, 88, 90, 127, or 129.

[0176] Tolerated variations in antigen-binding domain sequences will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NOs:96, 98, 99, 100, 103, 105, 107, 109, 129, or 131.

[0177] The antigen-binding domain can include any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. In some embodiments, the portion of the antigen-binding domain comprises a mammalian antibody or a fragment thereof. The choice of antigen-binding domain can 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-chain 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 term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin (e.g., murine 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 peptide encoding a linker, which connects the N-terminus of the VH to the C-terminus of the VL, or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., an IL13Rα2-binding domain) comprises an scFv with a VH-linker-VL configuration from N-terminus to C-terminus. In some embodiments, the antigen-binding domain comprises an scFv with 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] The linker is usually rich in glycine for flexibility, and serine or threonine for solubility. The linker can connect 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 WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Glycine-serine (GS) linkers, such as (GS) n , (GSGGS) n (SEQ ID NO: 148), (GGGS) n (SEQ ID NO: 149), and (GGGGS) nA variety of linker sequences are known in the art, including, but not limited to, (SEQ ID NO:150) (wherein n represents an integer of at least 1). Exemplary linker sequences can comprise amino acid sequences, including, but not limited to, 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), GGGSGGGGSGGGGS (SEQ ID NO:158), and the like. One of skill 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 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are linked by the nucleic acid sequence The fragments are separated by a linker sequence having the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 158), which can be coded for by TIFF0007748935000001.tif12148.

[0181] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. 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. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity 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 et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., 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 the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain yields two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0183] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, resulting in two antigen-binding (ab') (bivalent) regions, each of which contains two separate amino acid chains, a portion of a heavy chain for antigen binding and a light (L) chain, linked by an S-linkage, 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 be derived from the same species as the species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen-binding domain may be derived from a species different from the species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may comprise a mouse antibody or a fragment thereof.

[0185] In some embodiments, the CAR of the present 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 or multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR comprising one or more target-specific binding domains with affinity for the same target antigen can also bind to distinct 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 a linker peptide. For example, in a CAR comprising two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain via an oligopeptide or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0186] Transmembrane domain The CAR of the present invention can comprise a transmembrane domain that connects the antigen binding domain of the CAR with the intracellular domain of the CAR. The transmembrane domain of the subject CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor cell). The transmembrane domain is for insertion into a cell membrane, for example, a 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 of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains with the transmembrane domains of the same or different surface membrane proteins, minimizing interaction with other members of the receptor complex.

[0188] Transmembrane domain can be derived from either natural or synthetic source.When source is natural, domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When source is synthetic, transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains of particular use in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane regions of) the alpha, beta, or zeta chains of the T-cell receptor, 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, or transmembrane domains derived from killer cell immunoglobulin-like domains (KIR). In one embodiment, the transmembrane domain comprises the transmembrane domain of CD8. In one embodiment, the transmembrane domain of CD8 comprises the transmembrane domain of CD8α.

[0189] In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet will be found at each end of a 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 of the other domains described herein that can be included in a subject CAR.

[0191] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise 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 proper protein folding for the CAR. The hinge region is an optional component of a CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, 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 can be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0192] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen-binding domain to the transmembrane domain, which in turn connects it to the intracellular domain. The hinge region preferably can assist the antigen-binding domain in recognizing and binding to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows the hinge region to adopt 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 about 4 amino acids to about 50 amino acids, e.g., about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0195] A suitable hinge region can be readily selected and can be any of several suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 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. A suitable hinge region can be 20 amino acids long (e.g., 30, 40, 50, 60, or more amino acids).

[0196] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 148) and (GGGS) n(SEQ ID NO:149), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the φ-ψ space than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions include, but are not limited to: It can contain amino acid sequences such as TIFF0007748935000002.tif13164.

[0197] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are 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, an immunoglobulin hinge region may have the following amino acid sequence: It can contain one of the following: TIFF0007748935000003.tif49159.

[0198] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be substituted with Tyr, thereby causing the hinge region to comprise the sequence EPKSCDKTYTCPPCP (SEQ ID NO:85); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0199] Intracellular domain The CAR of the present invention also comprises an intracellular domain. In some embodiments, the intracellular domain comprises a costimulatory 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 (e.g., immune cell) in which the CAR is expressed. The intracellular domain transmits an effector function signal, directing the cell (e.g., immune cell) to perform its specialized function, such as damaging and / or destroying target cells.

[0200] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecules that act in concert to initiate signaling 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, such as the eta chain, FcsRI gamma and beta chains, MB1 (Iga) chain, B29 (Ig) chain, etc., 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 aspect, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, and combinations thereof.

[0202] In certain embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, for example, 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 capability, and any combination thereof. The intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in 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 comprises the costimulatory domain of 4-1BB.

[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 RIa, 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, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include fragments or domains derived from one or more molecules or receptors, including, but 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 derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0204] Additional examples of intracellular domains include the intracellular signaling domains of several different immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of 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.

[0205] In certain embodiments, the intracellular domain comprises an intracellular signaling domain selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof. In certain embodiments, the intracellular domain comprises the intracellular domain of CD3ζ.

[0206] Intracellular domains suitable for use in the subject CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; changes in transcription of target genes; changes in protein activity; changes in cellular behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses, etc.) in response to CAR activation (i.e., activated by an antigen and a dimerizing agent). 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 not covalently linked to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0207] Intracellular domains suitable for use in the subject CARs of the present invention comprise an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice within the intracellular domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular domain of a subject CAR comprises three ITAM motifs.

[0208] In some embodiments, the intracellular domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0209] A suitable intracellular domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular domain does not need to contain the entire sequence of the entire protein from which it is derived. 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 aspect, the intracellular domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer-activating receptor-associated protein; killer-activating receptor-associated protein, etc.). In one aspect, the intracellular domain is derived from FCER1G (also known as 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; immunoglobulin E receptor, high-affinity gamma chain, etc.). In one aspect, the intracellular domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, 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 aspect, the intracellular domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular domain is derived from CD79A (B cell antigen receptor complex-associated protein alpha chain; also known as 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 the present disclosure comprises a DAP10 / CD28-type signaling chain. In one embodiment, the intracellular domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular domain comprises the 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 the cytoplasmic signaling domain of human CD3 zeta.

[0211] Typically, the entire intracellular domain can be used, although in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits the effector function signal. The intracellular domain includes any truncated portion of the intracellular domain sufficient to transmit the effector function signal.

[0212] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0213] Table 1: Sequences used in the present invention TIFF0007748935000004.tif70157TIFF0007748935000005.tif245157TIFF0007748935000006.tif244157TIFF0007748935000007.tif243157TIFF0007748935000008.tif247157TIFF0007748935000009.tif247157TIFF0007748935000010.tif243157TIFF0007748935000011.tif244157TIFF0007748935000012.tif243157TIFF0007748935000013.tif243157TIFF0007748935000014.tif243157TIFF0007748935000015.tif243157TIFF0007748935000016.tif244157TIFF0007748935000017.tif243157TIFF0007748935000018.tif242157TIFF0007748935000019.tif245157TIFF0007748935000020.tif247157TIFF0007748935000021.tif243157TIFF0007748935000022.tif243157TIFF0007748935000023.tif247157TIFF0007748935000024.tif243157TIFF0007748935000025.tif243157TIFF0007748935000026.tif243157TIFF0007748935000027.tif150157

[0214] C. Tandem bispecific CARs and parallel bispecific CARs Also provided herein are tandem CARs, cells (e.g., T cells) comprising tandem CARs, amino acid sequences comprising tandem CARs, and nucleic acids encoding tandem CARs. Tandem CARs comprise 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) followed by a second antigen-binding domain (e.g., a second scFv) separated by a linker, which comprises 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 comprising an scFv capable of binding to IL13Rα2, and a second antigen-binding domain comprising an scFv capable of binding to EGFR.

[0215] The linker in a tandem CAR that connects the first and second antigen-binding domains can be of various sizes, e.g., any number of amino acids in length (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 acids in length. In certain embodiments, a tandem CAR comprises a linker that is 5 amino acids in length. In certain embodiments, a tandem CAR comprises the amino acid sequence of SEQ ID NO: 163 and can be encoded by the nucleotide sequence of SEQ ID NO: 164. In certain embodiments, a tandem CAR comprises a linker that is 10 amino acids in length. In certain embodiments, a tandem CAR comprises the amino acid sequence of SEQ ID NO: 165 and can be encoded by the nucleotide sequence of SEQ ID NO: 166. In certain embodiments, the tandem CAR comprises a linker that is 15 amino acids in length. In certain embodiments, the tandem CAR comprises the amino acid sequence of SEQ ID NO: 167 and can be encoded by the nucleotide sequence of SEQ ID NO: 168.

[0216] Also provided herein are parallel CARs, cells (e.g., T cells) comprising parallel CARs, amino acid sequences comprising parallel CARs, and nucleic acids encoding parallel CARs. Parallel CARs comprise 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., an scFv) linked to a first transmembrane domain and a first intracellular domain, a cleavable linker (e.g., a 2A linker), and a second antigen-binding domain (e.g., an scFv) linked to a second transmembrane domain and a second intracellular domain. When the nucleic acid is expressed in a cell, 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 the amino acid sequence of SEQ ID NO: 171 and can be encoded by the nucleotide sequence of SEQ ID NO: 172.

[0217] D. BiTE, BiTE / BiTE, and BiTE / CAR combinations Bispecific T cell engagers (BiTEs) and BiTE / CAR combinations are provided herein. The BiTEs comprise a first antigen-binding domain (e.g., a first scFv) and a second antigen-binding domain (e.g., a second scFv), where 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 on an activated T cell (e.g., CD3, CD4, CD8, or TCR).

[0218] In one aspect, the invention includes a BiTE capable of binding to IL13Rα2. In one aspect, the invention includes a BiTE capable of binding to CD3 and IL13Rα2. In certain embodiments, a BiTE comprises any of the antigen-binding domains disclosed herein capable of binding to IL13Rα2. In certain embodiments, a BiTE comprises an antigen-binding domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-22.

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

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

[0221] Also provided herein are bispecific constructs comprising 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).

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

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

[0224] A BiTE / BiTE can 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 an isoform thereof, and a second BiTE capable of binding to IL13Rα2. In certain embodiments, a BiTE / BiTE comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:179, which can be encoded by a nucleotide sequence 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 The present disclosure provides nucleic acids encoding a CAR. The nucleic acids of the present disclosure can include a polynucleotide sequence encoding any one of the CARs, BiTEs, BiTE / CARs, or BiTEs / BiTEs disclosed herein.

[0226] In one aspect, a nucleic acid of the disclosure comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) capable of binding to IL13Ra2, 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), wherein HCDR1 comprises the amino acid sequence TKYGVH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence GVKWAGGSTDYNSALMS (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).

[0227] In one embodiment, the nucleic acid encodes a CAR comprising 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.

[0228] In one embodiment, the nucleic acid encodes a CAR comprising an antigen-binding domain comprising 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 comprising 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 that is a single chain 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), 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 QGTTALATRFFDV (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).

[0232] In one embodiment, the nucleic acid encodes a CAR comprising 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:67.

[0233] In one embodiment, the nucleic acid encodes a CAR comprising an antigen-binding domain comprising 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 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: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 that is a single chain 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 a nucleic acid 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 an isoform thereof, wherein the first CAR and the second CAR each comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0240] In certain embodiments, 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 certain embodiments, 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 certain embodiments, 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 / 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 certain embodiments, 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 / 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 certain embodiments, the antigen-binding domain of the first CAR is a single-chain 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 SEQ ID NO:133 or SEQ ID NO:134 or SEQ ID NO:135.

[0245] In certain embodiments, 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.

[0246] In certain embodiments, 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 certain embodiments, the antigen binding domain of the second 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: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 certain embodiments, the antigen binding domain of the second 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:144 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:146. In certain embodiments, the antigen binding domain of the second 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:145 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:147.

[0248] In certain embodiments, the antigen binding domain of the second 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:42 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:43.

[0249] In certain embodiments, the antigen binding domain of the second CAR is a single-chain 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 SEQ ID NO:141 or SEQ ID NO:41. In certain embodiments, the antigen binding domain of the second CAR is a single-chain 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 or SEQ ID NO:142 or SEQ ID NO:44.

[0250] 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 SEQ ID NO:35 or 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 or SEQ ID NO:38 or SEQ ID NO:197.

[0251] 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 an isoform thereof, wherein the first CAR has 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:5). 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) or KASQDVGTAVA (SEQ ID NO:16), LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:6) or SASYRST (SEQ ID NO:17), and LCDR3 comprises the amino acid sequence HQYHRSPLT (SEQ ID NO:7) or QHHYSAPWT (SEQ ID NO:18); a second CAR comprising 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:19). 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).

[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 an isoform thereof, 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: 62, or 73. 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 an isoform thereof, wherein the first CAR comprises a single-chain 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-chain 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 an isoform thereof, 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 includes nucleic acids 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 are nucleic acids 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 isoforms. 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 a 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 present disclosure provide for the production of a CAR described herein, e.g., in a mammalian cell. In some embodiments, the nucleic acids of the present disclosure provide for the amplification of a nucleic acid encoding a CAR.

[0258] In some embodiments, a nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first polynucleotide sequence and the second polynucleotide sequence may be separated by a linker. A linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence) and translated as a polyprotein that dissociates into separate protein components. For example, a linker for use in a nucleic acid of the present disclosure comprising a coding sequence for an IL13Ra2 CAR and a coding sequence for an EGFR CAR allows the IL13Ra2 CAR and the EGFR CAR to be translated as a polyprotein that dissociates into separate CARs. In certain embodiments, a nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, a nucleic acid comprises, from 5' to 3', a second polynucleotide sequence, a linker, and a first polynucleotide sequence.

[0259] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal 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 internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs 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 IRESs available from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select a suitable IRES for use in the present invention.

[0260] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows encoding of 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. Various self-cleaving or 2A peptides are known to those of skill 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 siagnavirus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those of skill in the art will be able to select a suitable self-cleaving peptide 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 that resides in the trans-Golgi and processes protein precursors prior to their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including, but not limited to, 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), where X1 is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0262] In some embodiments, the linker comprises 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 comprising a nucleic acid sequence encoding furin and F2A, a linker comprising a nucleic acid sequence encoding furin and E2A, a linker comprising a nucleic acid sequence encoding furin and P2A, or a linker comprising 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 comprise a spacer sequence between furin and the 2A peptide. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO:148), and (GGGS)n (SEQ ID NO:149), where n represents an integer of at least 1. Exemplary spacer sequences include, but are not limited to: TIFF0007748935000028.tif20158, etc. Those skilled in the art will be able to select suitable spacer sequences for use in the present invention.

[0263] In some aspects, the nucleic acids of the present disclosure may be operably linked to transcriptional control elements, such as promoters, enhancers, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

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

[0265] Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. Suitable promoters for expression in eukaryotic cells include, but are not limited to, the light and / or heavy chain immunoglobulin gene promoter and enhancer element; the cytomegalovirus immediate early promoter; the herpes simplex virus thymidine kinase promoter; the early and late SV40 promoter; the promoter present in the long terminal repeat of retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. The modification of a reversible promoter 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.) is 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-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated 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-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0266] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, 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, PYK1 promoter, etc.; or regulatable 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 (e.g., for use in Pichia). Selection of an appropriate vector and promoter is well within the level of one skilled in the art. Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the 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), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, e.g., consensus sigma70 promoters (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters, e.g., dps promoters, spv promoters, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted 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 immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence operably linked to it when such expression is desired, or turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In certain embodiments, the present invention provides a polynucleotide sequence encoding a CAR (e.g., a bispecific CAR, a BiTE, a tandem CAR, a parallel CAR, and the like) comprising an inducible promoter. In certain embodiments, the inducible promoter promotes expression of an operably linked sequence (e.g., a CAR) after T cell activation. T cells (e.g., CAR T cells) can be engineered with this promoter to express a designed RNA or amino acid. In certain embodiments, the inducible 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 comprises 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, a sequence comprising SEQ ID NO: 198 is repeated to enhance expression levels in T cells. For example, in certain embodiments, the inducible promoter can comprise 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 inducible system. Suitable systems for inducing irreversible switches are well known in the art; for example, induction of irreversible switches may utilize Cre-lox-mediated recombination (see, e.g., 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, and the like known in the art may be used to generate irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, for example, Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0270] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducing expression cassette. In one embodiment, the CAR-inducing expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, e.g., 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 acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T cell activation response promoter. In some embodiments, the cytokine operably linked to the T cell activation response promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0271] The nucleic acids of the present disclosure can be present in an expression vector and / or a cloning vector. Expression vectors can include a selectable marker, an origin of replication, and other features that provide for 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 of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example and 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). Prokaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0272] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. 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: 1088-1097; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (see, 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 in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, 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., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like.

[0273] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. 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. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0274] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0275] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce a CAR into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding a CAR. In some embodiments, an expression vector (e.g., a lentiviral vector) will comprise additional elements that aid in the functional expression of a CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a CAR). A physiological promoter (e.g., an EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector 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 vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-essential cis-acting sequence that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present invention. In some embodiments, the vector further comprises a post-transcriptional regulatory element. 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 comprises a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present invention.The vectors of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a base-pair domain located at the end of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors of the present invention (e.g., lentiviral vectors) comprise a 3' U3-deleted LTR. Thus, the vectors of the present invention (e.g., lentiviral vectors) may comprise any combination of the elements described herein to increase the efficiency of functional expression of the transgene. For example, the vectors of the present invention (e.g., lentiviral vectors) may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, and a 3' U3-deleted LTR' in addition to the nucleic acid encoding a CAR.

[0276] The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3' LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then be integrated into the host genome (e.g., a mammalian genome) only once, but cannot be further passaged. Therefore, a self-inactivating vector can greatly reduce the risk of generating a replication-competent virus.

[0277] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into host cells can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure in vitro or ex vivo.

[0278] To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population to be transfected or infected with a viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be adjacent to appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0279] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and encode polypeptides whose expression is revealed by some easily detectable characteristics, such as enzymatic activity.The expression of reporter genes is evaluated at an appropriate time after DNA is introduced into recipient cells.Suitable reporter genes may include, but are not limited to, luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

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

[0281] One aspect of the present invention provides an engineered immune cell or progenitor thereof comprising a CAR capable of binding to IL13Rα2, wherein the 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 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).

[0282] Another aspect of the invention includes an engineered immune cell or a precursor thereof comprising a CAR capable of binding to IL13Rα2, wherein the CAR comprises a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20.

[0283] Also provided are engineered immune cells or precursor cells thereof comprising a CAR capable of binding to IL13Rα2, wherein the CAR comprises a single chain 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.

[0284] In another aspect, the present invention provides an engineered immune cell or progenitor thereof comprising a 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.

[0285] Another aspect of the present invention includes an engineered immune cell, or a precursor thereof, 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 an isoform thereof.

[0286] Yet another aspect of the present invention includes an engineered immune cell or progenitor thereof comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, 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 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).

[0287] Yet another aspect of the invention includes an engineered immune cell or progenitor thereof comprising a first CAR capable of binding to IL13Rα2 and a second CAR capable of binding to epidermal growth factor receptor (EGFR) or an isoform thereof, wherein the first CAR comprises a heavy chain variable region comprising 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 comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or 20. The second CAR 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:31 or SEQ ID NO:42 or SEQ ID NO:144 or SEQ ID NO:145, 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 or SEQ ID NO:43 or SEQ ID NO:146 or SEQ ID NO:147.

[0288] Also provided is an engineered immune cell or progenitor 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 a single-chain 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 SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:22; and the second CAR comprises a single-chain 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 or SEQ ID NO:44.

[0289] In certain embodiments, the second CAR is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, and EGFR. A289V , EGFR A289D , EGFR A289T , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F and EGFR variant II, or any combination thereof.

[0290] The modified cells can further comprise an immune checkpoint inhibitor, wherein the modified cells secrete the immune checkpoint inhibitor. 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 engineered cells can further comprise an inducible bispecific T cell engager (BiTE) capable of binding to epidermal growth factor receptor (EGFR) or its isoforms. The engineered cells secrete the BiTE. In certain embodiments, the inducible BiTE comprises an amino acid sequence 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 a subject. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells can be derived from the subject to be treated with the modified immune cells of the present invention, and can 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 transgenic species thereof. 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, for example, lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, for example, primary cells isolated directly from the subject and / or isolated and frozen from the subject.

[0294] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In certain embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce mutations in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0295] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturation, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4+ and / or CD8+ T cell subtypes and subpopulations include, among others, naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, innate and adaptive regulatory T (Treg) cells, helper T cells, such as 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, any number of T cell lines available in the art may be used.

[0296] In some embodiments, the method includes isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, preparing the engineered cells includes one or more culturing 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 or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of or to whom cell therapy will be administered. The subject, in some embodiments, is a human in need of a specific therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells, in some embodiments, 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 can 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, tissue and organ samples, including processed samples derived therefrom.

[0297] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0298] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from heterologous sources, e.g., mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells includes one or more preparative steps and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, enrich for 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, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0299] In some examples, cells from a subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in a buffer or medium suitable for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed, and the cells are resuspended directly in culture medium. In some embodiments, the method includes a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0300] In one embodiment, the immune cells are cells obtained from an individual's circulating blood by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium, such as phosphate-buffered saline (PBS). Alternatively, the wash solution may be calcium-free and magnesium-free, or may lack 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 culture medium.

[0301] In some embodiments, the isolation method involves separating different cell types based on the expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in cells. In some embodiments, any known method can be used for such marker-based separation. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the separation involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step, and separating the cells that bind to the antibody or binding partner from the cells that do not bind to the antibody or binding partner.

[0302] Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which cells that are not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, 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 necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0303] In some instances, multiple rounds of separation steps are performed, with the positively or negatively selected fraction from one step being subjected to another separation step, such as a subsequent positive or negative selection. In some instances, cells co-expressing multiple markers can be depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

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

[0305] In some embodiments, T cells are separated from PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate 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 on or expressed at a relatively high level 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, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0306] In some embodiments, memory T cells exist in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions using, for example, anti-CD8 and anti-CD62L antibodies. In some embodiments, the CD4+ T cell population and CD8+ T cell subpopulation, e.g., a subpopulation enriched for central memory (TCM) cells, are enriched for central memory T (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed 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, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in other aspects, in either order. In some aspects, the same selection step based on CD4 expression used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, whereby both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0307] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations bearing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In 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 antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to enable separation of cells for positive and / or negative selection.

[0308] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory substance. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. Conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the stimulatory conditions or stimulatory substances include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of a TCR complex. In some aspects, the agent triggers or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such an agent can include, for example, an antibody bound to a solid support such as a bead, e.g., an antibody specific for a TCR component and / or a costimulatory receptor, e.g., anti-CD3, anti-CD28, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulator includes IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL.

[0309] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cords. In any event, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0310] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, or antibodies bound to cells.

[0311] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0312] To isolate a desired population of cells by positive or negative selection, cell concentration and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which b...

Claims

1. A nucleic acid comprising: a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) capable of binding to IL13Rα2, the first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising an IL13Rα2 antigen-binding domain, a transmembrane domain, and an intracellular domain; and a second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) capable of binding to epidermal growth factor receptor (EGFR), the second polynucleotide sequence encoding a second chimeric antigen receptor (CAR) comprising an EGFR antigen-binding domain, a transmembrane domain, and an intracellular domain; (a) The first CAR is: (i) 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 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); contains, or (ii) 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 QGTTALATRFFDV (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 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); and (b) 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 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); A nucleic acid comprising:

2. (a) The first CAR is: (i) a heavy chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 57 and a light chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 61; or (ii) a heavy chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 67 and a light chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 71 and (b) The second CAR is (i) a heavy chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 139 and a light chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 140; or (ii) a heavy chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 194 and a light chain variable region encoded by the polynucleotide sequence of SEQ ID NO: 195 The nucleic acid of claim 1, comprising:

3. (a) the first CAR comprises a single-chain variable fragment (scFv) encoded by the polynucleotide sequence of SEQ ID NO: 133, 134, 135, or 138; and (b) the second CAR comprises a single-chain variable fragment (scFv) encoded by the polynucleotide sequence of SEQ ID NO: 33 or 141; 3. The nucleic acid of claim 1 or 2.

4. (a) the first polynucleotide sequence comprises the sequence of SEQ ID NO:65 or SEQ ID NO:66 or SEQ ID NO:75 or SEQ ID NO:76; and (b) the second polynucleotide sequence comprises the sequence of SEQ ID NO: 35 or SEQ ID NO: 196; The nucleic acid of claim 1.

5. (a) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker; and / or (b) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker comprising a nucleotide sequence encoding an internal ribosome entry site (IRES) or a self-cleaving peptide; and / or (c) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, and the linker comprises a self-cleaving peptide that is a 2A peptide; and / or (d) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, and the linker comprises a self-cleaving peptide that is a 2A peptide, and the 2A peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), Equine rhinitis A virus 2A (E2A), and Foot-and-Mouth Disease virus 2A (F2A); (e) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker comprising a self-cleaving peptide that is a 2A peptide, and the 2A peptide is T2A; and / or (f) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker further comprising a furin cleavage site; and / or (g) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, and the nucleic acid comprises, in a 5' to 3' direction, the first polynucleotide sequence, the linker, and the second polynucleotide sequence; and / or (h) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, and the nucleic acid comprises, in a 5' to 3' direction, the second polynucleotide sequence, the linker, and the first polynucleotide sequence; A nucleic acid according to any one of claims 1 to 4.

6. 6. The nucleic acid of any one of claims 1 to 5, wherein the nucleic acid further comprises an inducible promoter, wherein the inducible promoter comprises the nucleotide sequence of SEQ ID NO: 161, 162, or 198.

7. A vector comprising the nucleic acid of any one of claims 1 to 6.

8. (a) is an expression vector; and / or (b) is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector; and / or (c) further comprising an EF-1a promoter; and / or (d) further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and / or (e) further comprising a rev response element (RRE); and / or (f) further comprising a cPPT sequence; and / or (g) a self-inactivating vector; The vector of claim 7.

9. 9. A modified immune cell or a precursor thereof comprising a nucleic acid according to any one of claims 1 to 6, or a vector according to claim 7 or 8.

10. (a) a first chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to IL13Rα2, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain; and (b) a second chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding to the epidermal growth factor receptor (EGFR), a transmembrane domain, a costimulatory domain, and an intracellular signaling domain; 1. A modified immune cell or a precursor thereof comprising: (a) The first CAR is: (i) 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 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); or (ii) 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 QGTTALATRFFDV (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 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). and (b) 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 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); 1. A modified immune cell or a precursor thereof, comprising:

11. (a) The first CAR is: (i) a heavy chain variable region comprising an amino acid sequence at least 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence at least 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9; or (ii) a heavy chain variable region comprising an amino acid sequence at least 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence at least 97%, 98%, 99%, or 100% identical to SEQ ID NO:

20. Including, (b) the second CAR comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; Including, The modified immune cell or precursor cell thereof of claim 10.

12. (a) a first CAR comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 10, 11, 21, or 22, and a second CAR comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 34, or (b) the first CAR comprises the amino acid sequence of SEQ ID NO: 23 or 24, and the second CAR comprises the amino acid sequence of SEQ ID NO: 36 or 197; The modified immune cell or precursor cell thereof of claim 10.

13. 13. The modified immune cell, or progenitor thereof, of any one of claims 10-12, wherein a first CAR is capable of binding to human IL13Rα2 and a second CAR is capable of binding to wild-type EGFR (wtEGFR) or EGFR variant III.

14. The modified immune cells or precursor cells thereof are (a) is an engineered T cell; and / or (b) are autologous; and / or (c) are autologous cells obtained from a human subject; 14. The modified immune cell or precursor thereof of any one of claims 10 to 13.

15. 15. A pharmaceutical composition comprising a therapeutically effective amount of the modified immune cells or precursor cells thereof of any one of claims 10 to 14.

16. 16. A composition comprising the modified immune cell or progenitor cell thereof of any one of claims 10 to 14, or the pharmaceutical composition of claim 15, for use in treating a disease in a subject in need thereof.

17. (a) the disease is cancer; and / or (b) the disease is cancer and the cancer is glioma; and / or (c) the disease is cancer and the cancer is astrocytoma; and / or (d) the disease is cancer and the cancer is high-grade astrocytoma; and / or (e) the disease is cancer, and the cancer is glioblastoma; 17. The composition of claim 16.

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